Blood storage container containing aqueous composition for the storage of red blood cells

ABSTRACT

A blood storage container along with an aqueous composition for the storage of packed red blood cells is described. In a preferred embodiment, the container is not made of DEHP plasticizer. In some embodiments, the container is made from a polymeric material and a non-DEHP plasticizer. In some embodiments, the aqueous composition is made of about 1 to about 3 mM adenine, about 20 to about 15 mM dextrose, about 15 to about 60 unmetabolizable membrane-protectant sugar, about 20 to about 30 mM sodium bicarbonate, and about 4 to about 20 mM disodium phosphate. In a most preferred embodiment, the DEHP-lacking container is made from a PVC polymeric material and a DINCH plasticizer and the aqueous composition is made of about 2 mM adenine, about 80 mM dextrose, 55 mM unmetabolizable membrane-protectant sugar, about 26 mM sodium bicarbonate, and about 12 mM disodium phosphate

PRIORITY

This patent application claims priority from U.S. Provisional PatentApplication Ser. No. 61/692,048, filed Aug. 22, 2012, entitled,“Non-DEHP Blood Storage Bag Containing Aqueous Composition for theStorage of Packed Red Blood Cells,” and naming Majid Zia as inventor,the disclosure of which is incorporated herein, in its entirety, byreference.

BACKGROUND OF THE DISCLOSURE

The present invention relates to compositions and methods associatedwith the storage of red blood cells (RBCs). In particular, it relates toan improved RBC storage composition in combination with a blood storagecontainer.

Red blood cells are often separated from other components of whole bloodfrom donors (e.g., platelets, white blood cells, and plasma) and arecollected for later transfusion to a patient in need of red blood cells.For example, red blood cells (hereinafter “RBCs”) may be administered toa patient suffering from a loss of blood due to trauma, as apost-chemotherapy treatment or surgery, or as part of a treatment of oneor more blood borne diseases, such as certain anemias and the like.Unless administered immediately after collection from a donor, RBCs musttypically be stored for some period of time prior to transfusion. Thestorage period may be anywhere from a few days to several weeks.

Prolonged storage of RBCs can (negatively) affect RBC function. In orderfor the RBCs to be suitable for transfusion to the recipient, RBCs mustmaintain adequate cell function and metabolism. For example, RBCs mustmaintain an adequate concentration of adenosine triphosphate (ATP). Inaddition, stored RBCs must have acceptably low levels of hemolysis.Typically, an acceptable level of hemolysis is below 1.0% (in, forexample, the U.S. with 95% confidence and 95% reliability) and below0.8% (in Europe with 90% of the tested units) after 42 day storage.

The ability to store and preserve red blood cells (RBCs) for laterre-infusion into patients is a relatively recent technologicaldevelopment that was the harbinger to modern surgical practice. Suchpreservation is scientifically tricky and the steps to achieving longerstorage duration and higher quality re-infused red blood cells have beenincremental. As soon as they are collected from a donor, red blood cellsbegin to die as they coagulate, starve, lose ATP, 2,3-DPG, membranesurface area and integrity, and hemoglobin (Hb). Rous & Turner in 1916and Robertson in 1917 15 first demonstrated successful whole bloodstorage. Acid-citrate-dextrose (ACD, 1943), comprising citrate as ananti-coagulant and dextrose as the sole nutrient utilized by red bloodcells, and Citrate-phosphate-dextrose solution (CPD, 1957), addingphosphate as a metabolic source and for membrane retention, weresubsequently approved for 21-day storage of whole blood. CPD withadenine (CPDA-1, 1979) was later introduced and used for extending theshelf life of stored whole blood and packed RBCs for up to 5 weeks.

Additive solutions for providing a storage environment for RBCs thatwill allow cell function and cell metabolism to be preserved andmaintained have been developed and are commonly used. The additivesolutions (i.e., media developed for RBCs) can prolong the storage lifeof RBCs for up to 42 days. These additive solutions often include anutrient for the RBCs, a buffer to help maintain the pH of the RBCs,electrolytes, a RBC membrane-protecting compound and other additives toenhance and extend the life of the RBCs.

Traditionally, packed RBCs are stored in PVC bags. During manufacture,the PVC is mixed with a plasticizer so that the bags may be moldedand/or welded into an appropriate shape. The PVC bags used in bloodstorage today are formulated with a plasticizer known as di-ethyhexylphthalate (DEHP), up to 30-40% by weight. Because DEHP is not bound tothe polymer in a PVC medical device, it is known to leach from the PVCinto the storage solution at concentrations of up to 650 mg/liter andactually has been shown to have very beneficial effects in maintainingthe viability and long-term storage of RBCs. The DEHP may actually bindto the RBCs, possibly preserving them and extending their shelf-life.DEHP's potential role in preserving red blood cells is an unintentionalresult: DEHP was not added to PVC to increase the shelf-life of bloodcells, rather it was a serendipitous discovery.

However, DEHP may be toxic to humans. Studies in humans have foundadverse health effects such as respiratory distress, choleostasis, andhistological abnormalities of the liver. Animal studies have shown awide range of toxic effects, especially to developing fetuses. Risks arehigher in potentially sensitive groups, such as neonate and chronicallyill individuals. High exposure patients include patients requiringextensive blood or blood product transfusions.

Thus, there is not only a need for prolonged storage of red blood cells,but also a need for long-term storage in non-DEHP containers. The desireto steer away from the use of DEHP in PVC used for medical devices is sogreat that European nations are actively regulating against its use.

Many alternative plasticizers have been proposed, but have certainshortcomings, such as prohibitive expense and lack of toxicologystudies. More importantly, because the DEHP actually aids in thepreservation of the stored RBCs, without DEHP, traditional storagesolutions do not perform as well as with other plasticizers. In fact,the performance of one of the most commonly used storage solutions inbags without DEHP does not meet current regulatory standards for 42 daysRBC storage. The main obstacle to approval of additive solutions innon-DEHP containers remains excess hemolysis observed with the use ofnon-DEHP containers. Thus, there is a critical need for newplasticizer/storage composition combinations that avoid the use of DEHPbut yet can still perform within regulatory standards to maintain RBCquality for the current storage period.

SUMMARY OF THE EMBODIMENTS

Accordingly, the present invention provides a novel combination of bloodstorage container and aqueous composition suitable for the storage andpreservation of collected red blood cells. In an attempt to find such acombination, the inventor surprisingly discovered aqueous compositioncombined in a non-DEHP containing container are suitable for storing andpreserving collected red blood cells 1 to 6° C.

In one aspect, the invention provides a combination product for storingred blood cells, the product comprising, consisting essentially or, orconsisting of a container comprising, consisting essentially of, orconsisting of a wall defining an interior chamber wherein at least aportion of the wall is made of polymeric material combined with anon-phthalate plasticizer; and an aqueous composition contained withinsaid chamber, said composition comprising, consisting essentially of, orconsisting of adenine; dextrose; at least one non-metabolizablemembrane-protectant sugar; and a pH buffering system, wherein the pHbuffering system consists of a combination of physiologically acceptablebuffering agents including at least one agent providing bicarbonateanions, at least one agent providing phosphate anions, and at least oneagent providing sodium cations, wherein the aqueous composition issubstantially free of exogenously derived chloride ions, wherein the pHbuffering system is present in an amount sufficient for the compositionto be operable to maintain a pH of a red blood cell (RBC) suspension towhich the composition is added at a value sufficient to establish andmaintain during a storage period a reaction equilibrium in the red bloodcell that favors glycolysis over synthesis of 2,3-diphosphoglycerat(DPG) from 1,3-DPG, thereby generating a net gain in adenosinetriphosphate (ATP) with respect to the reaction equilibrium during thestorage period. In some embodiments, adenine is present in an amount ofbetween about 1 mM and about 3 mM. In some embodiments, dextrose ispresent in an amount of between about 20 mM and about 115 mM. In someembodiments, the unmetabolizable membrane-protectant sugar is present inan amount of between about 15 mM and about 60 mM. In some embodiments,the at least one agent providing bicarbonate anions is sodiumbicarbonate. In some embodiments, the at least one agent providingphosphate anions is disodium phosphate.

In another aspect, the invention provides a combination product forstoring red blood cells, the product comprising, consisting essentiallyor, or consisting of a container comprising, consisting essentially of,or consisting of a wall defining an interior chamber wherein at least aportion of the wall is made of polymeric material combined with anon-phthalate plasticizer; and an aqueous composition contained withinsaid chamber, said composition comprising, consisting essentially of, orconsisting of adenine in an amount of about 1-3 mM, dextrose in anamount of from about 20 to about 115 mM, disodium phosphate in an amountof from about 4 to about 20 mM; at least one unmetabolizablemembrane-protectant sugar in an amount of about 15 to about 60 mM, and aphysiologically acceptable sodium salt at about 20 mM to about 130 mM.In various embodiments, the sodium acceptable salt is sodiumbicarbonate. In some embodiments, the aqueous composition issubstantially free of exogenously derived chloride ions.

In yet another aspect, the invention provides a combination product forstoring red blood cells, the product comprising, consisting essentiallyor, or consisting of a container comprising, consisting essentially of,or consisting of a wall defining an interior chamber wherein at least aportion of the wall is made of polymeric material combined with anon-phthalate plasticizer; and an aqueous composition contained withinsaid chamber, said composition comprising, consisting essentially of, orconsisting of adenine in an amount of about 1-3 mM, dextrose in anamount of from about 20 to about 115 mM, unmetabolizablemembrane-protectant sugar in an amount of about 15 to about 60 mM,sodium bicarbonate in an amount from about 20 to about 130 mM, anddisodium phosphate in an amount of from about 4 to about 20 mM. In someembodiments, the aqueous composition is substantially free ofexogenously derived chloride ions.

In various embodiments, the combination product provides benefits bothin terms of the integrity and physiological functioning quality of thestored red blood cells with hemolysis levels required under regulatorylaw. The improved integrity and physiological functioning quality of thestored RBCs is expected when the stored RBCs are re-infused into thedonor (or other patient in need of a transfusion).

In one specific embodiment, the non-DEHP plasticizer is diisononylcyclohexane-1,2-dicarboxylate (DINCH). In some embodiments, thepolymeric material comprises polyvinyl chloride. In some embodiments,the polymeric material comprises a non-PVC composition. In someembodiments, the non-phthalate plasticizer is 1,2-cyclohexanedicaroxylic acid diisononyl ester. In some embodiments, thenon-phthalate plasticizer is non-DEHP plasticizer. In some embodiments,the non-phthalate plasticizer is butyl-n-trihexyl-citrate (BTHC);citrate ester acetyltri-nbutyl citrate (ATBC); di, (2, ethyl, hexyl)terephthalate (DENT); tri, (2-ethyl hexyl)trimellitate (TEHTM); ordiisononyl cyclohexane-1,2-dicarboxylate (Hexamoll/DINCH).

In some embodiments, red blood cells stored in the product have ahemolysis level that is below 1.0% when the storage period at about 1 to6° C. is at least 6 weeks. In some embodiments, red blood cells storedin the product have a hemolysis level that is below 1.0% when thestorage period at about 1 to 6° C. is at least 8 weeks. In someembodiments, red blood cells stored in the product have a hemolysislevel that is below 1.0% when the storage period at about 1 to 6° C. isat least 10 weeks. In some embodiments, red blood cells stored in theproduct have a hemolysis level that is below 1.0% when the storageperiod at about 1 to 6° C. is at least 12 weeks.

In some embodiments, the composition is substantially free of citrate.

In some embodiments, adenine is present in an amount of about 2 mM. Insome embodiments, dextrose is present in an amount of between about 60mM to about 100 mM. In some embodiments, dextrose is present in anamount of about 80 mM. In some embodiments, the unmetabolizablemembrane-protectant sugar is present in an amount of between about 40 mMand about 60 mM. In some embodiments, the unmetabolizablemembrane-protectant sugar is present in an amount of about 55 mM. Insome embodiments, the unmetabolizable membrane-protectant sugar ismannitol.

In some embodiments, wherein the sodium bicarbonate is present in anamount of between about 20 mM and about 130 mM or between about 20 mMand about 40 mM. In some embodiments, the sodium bicarbonate is presentin an amount of about 26 mM. In some embodiments, the disodium phosphateis present in an amount of between about 4 mM and about 20 mM or betweenabout 7 mM and about 15 mM. In some embodiments, the disodium phosphateis present in an amount of about 12 mM.

In some embodiments, the osmolarity of the composition is between about210 mOsmoles/liter and about 340 mOsmoles/liter. In some embodiments,the osmolarity of the composition is between about 220 mOsmoles/literand about 310 mOsmoles/liter.

In some embodiments, the aqueous composition comprises, consistsessentially of, or consists of adenine in an amount of about 2 mM,dextrose in an amount of about 80 mM, unmetabolizablemembrane-protectant sugar in an amount of about 55 mM, sodiumbicarbonate in an amount of about 26 mM, and disodium phosphate in anamount of about 12 mM, and that provides benefits both in terms of theintegrity and physiological functioning quality of the stored andhemolysis levels required under regulatory law for licensing, even whenused in combination with a non-DEHP plasticizer-containing PVC bloodstorage. The improved integrity and physiological functioning quality ofthe stored RBCs is expected when the stored RBCs are re-infused into thedonor (or other patient in need of a transfusion). In one specificembodiment, the non-DEHP plasticizer is diisononylcyclohexane-1,2-dicarboxylate (DINCH).

Also provided are methods for the storage of RBCs using the combinationof non-DEHP blood storage solutions and the experimental composition.The method of preserving red blood cells (RBCs) for a storage periodcomprises; (a) collecting a sample of whole blood containing the RBCs tobe stored and plasma in a blood storage container, wherein the bloodstorage container comprises either PVC or a non-PVC polymeric materialand a non-pthalate or a non-DEHP plasticizer, (b) mixing the sample ofcollected whole blood with an anticoagulant solution (e.g., CPD),thereby forming a suspension of collected whole blood; (c) treating thesuspension of collected whole blood to deplete the plasma andconcentrate the RBCs, thereby forming packed RBCs (either with orwithout reducing the leukocyte content of the whole blood or the redblood cells); (d) mixing the packed RBCs with an amount of an aqueouscomposition sufficient to form a suspension of RBCs having about 35% toabout 80% RBCs by volume; (e) cooling the suspension of RBCs to about 1to about 6° C.; and (f) storing the cooled suspension of RBCs accordingto standard blood bank procedures.

In some embodiments, the RBCs are stored for 6 weeks (i.e., 42 days), orstored for 8 weeks (i.e., 56 days), or stored for 10 weeks (i.e., 70days), or stored for 12 weeks (i.e., 84 days). In some embodiments, theaqueous composition comprises, consists essentially of, or consists ofadenine in an amount of about 1-3 mM, dextrose in an amount of fromabout 20 to about 115 mM, unmetabolizable membrane-protectant sugar inan amount of about 15 to about 60 mM, sodium bicarbonate in an amountfrom about 20 to about 130 mM, and disodium phosphate in an amount offrom about 4 to about 20 mM. In some embodiments, the aqueouscomposition comprises, consists essentially of, or consists of adenine;dextrose; at least one non-metabolizable membrane-protectant sugar; anda pH buffering system, wherein the pH buffering system consists of acombination of physiologically acceptable buffering agents including atleast one agent providing bicarbonate anions, at least one agentproviding phosphate anions, and at least one agent providing sodiumcations, wherein the aqueous composition is substantially free ofexogenously derived chloride ions, wherein the pH buffering system ispresent in an amount sufficient for the composition to be operable tomaintain a pH of a red blood cell (RBC) suspension to which thecomposition is added at a value sufficient to establish and maintainduring a storage period a reaction equilibrium in the red blood cellthat favors glycolysis over synthesis of 2,3-diphosphoglycerat (DPG)from 1,3-DPG, thereby generating a net gain in adenosine tri phosphate(ATP) with respect to the reaction equilibrium during the storageperiod.

In some embodiments, steps (a)-(c) of the above method are modified bycollecting the whole blood into CPD (e.g., 142 mM dextrose, 104 mMNa3Citrate, 18 mM NaH2PO4) or CP2D (e.g., 284 mM dextrose, 104 mMNa3Citrate, 18 mM NaH2PO4) and then leukoreducing the whole blood (e.g.,by passage through a filter or centrifuging) to obtain leukoreducedwhole blood. The leukoreduced whole blood is then centrifuged toseparate plasma from the packed red blood cells. The packed red bloodcells are mixed with the aqueous composition in the blood storagecontainer comprises PVC and a non-DEHP plasticizer and stored at about 1to about 6° C. according to standard blood bank procedures. In someembodiments, the RBCs are stored for 6 weeks (i.e., 42 days), or storedfor 8 weeks (i.e., 56 days), or stored for 10 weeks (i.e., 70 days), orstored for 12 weeks (i.e., 84 days).

In some embodiments, whole blood is collected in anticoagulant (e.g.,CPD, CP2D, or ACD), spun in a centrifuge and/or separated into variouscomponents including red blood cells which are collected into additivesolution (e.g., comprising, consisting essentially of, or consisting ofadenine; dextrose; at least one non-metabolizable membrane-protectantsugar; and a pH buffering system, wherein the pH buffering systemconsists of a combination of physiologically acceptable buffering agentsincluding at least one agent providing bicarbonate anions, at least oneagent providing phosphate anions, and at least one agent providingsodium cations, wherein the aqueous composition optionally issubstantially free of exogenously derived chloride ions). Red blood cellin additive solution is leukocyte reduced and stored for the storageperiod.

In some embodiment, additive solution (e.g., comprising, consistingessentially of, or consisting of adenine; dextrose; at least onenon-metabolizable membrane-protectant sugar; and a pH buffering system,wherein the pH buffering system consists of a combination ofphysiologically acceptable buffering agents including at least one agentproviding bicarbonate anions, at least one agent providing phosphateanions, and at least one agent providing sodium cations, wherein theaqueous composition is substantially free of exogenously derivedchloride ions) is added to anticoagulated blood collected viaerythrocytapheresis and stored at 1 to 6 C.

These and additional embodiments and aspects of the present inventionwill be more fully appreciated by reference to the brief description ofthe figures, detailed description of the preferred embodiments andexamples provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features of embodiments will be more readily understood byreference to the following detailed description, taken with reference tothe accompanying drawings, in which:

FIG. 1 is a graphical depiction of paired outcomes at day 42 of storageof red blood cells in AS-5 (Optisol) additive solution. Part A showsmixed (Yes—on right) and non-mixed (No—on left) RBCs stored in DINCH-PVCcontainers for percent hemolysis, extracellular potassium, and ATP(umol/g-Hb). Part B shows unmixed RBCs stored in DEHP-PVC containers(left) compared with mixed RBCs (weekly mixed) stored in DINCH-PVCcontainers (right). Data from Dr. Larry Dumont presented at AmericanSociety of Haematology 2009, poster presentation.

FIG. 2 is a graphical depiction of standardized mean hemolysis ratios ofred blood cells stored in a storage composition consisting essentiallyof adenine in an amount of about 1-3 mM, dextrose in an amount of fromabout 20 to about 115 mM, unmetabolizable membrane-protectant sugar inan amount of about 15 to about 60 mM, sodium bicarbonate in an amountfrom about 20 to about 130 mM, and disodium phosphate in an amount offrom about 4 to about 20 mM for 42 days, stored in either DEHP, DINCH,or BTHC bags as compared to red blood cells stored in DEHP bags at 5, 6,7, 8, or 9 weeks of storage. As these are ratios of compared to the DEHPbags, the DEHP bag (black bars) is 1.0. The middle medium gray bar isthe DINCH bag while the right light gray bar is the BTHC bag.

FIG. 3 is a bar graph showing the morphology scores of RBCs stored for42 days stored in the AS-7 solution in DINCH containers or AS-1 in DEHPcontainers.

FIG. 4 is a bar graph showing the microvesicle protein level of RBCsstored for 43 days stored in the experimental composition in DINCHcontainers or AS-1 in DEHP containers.

FIG. 5 is a bar graph showing the level of ATP (expressed as a percentof initial ATP level) of RBCs stored for 42 days stored in theexperimental composition in DINCH containers or AS-1 in DEHP containers.

FIGS. 6A and 6B are a bar graph and a line graph, respectively, showingthe amount of hemolysis (measured as percent hemoglobin content) of RBCsstored for 42 days stored in the experimental composition in DINCHcontainers or AS-1 in DEHP containers. FIG. 6A shows the mean of the tenpools, FIG. 6B shows the individual pools.

FIG. 7 is a bar graph showing the residual white blood cell (WBC) countof RBC units in SOLX in DINCH containers or in AS-1 in DEHP containersthat will be stored (this is at day 0).

FIG. 8 is a bar graph showing the residual hemoglobin content of RBCspost filtration (i.e., filtration to reduce white blood cells) in SOLXin DINCH containers or in AS-1 in DEHP containers that will be stored(this is at day 0).

FIG. 9 is a bar graph showing the ratio of the post-filtration of RBCmass divided by pre-filtration RBC mass of whole blood units that willbe processed into packed RBCs and then stored in SOLX/DINCH containersor in AS-1 in DEHP containers. The filtration removes (by trapping)white blood cells and also traps some red blood cells.

FIG. 10 is a bar graph showing the unit volume of whole blood units inCPD pre-filtration (i.e., prior to filtration), RBCs of which willeither be stored in SOLX/DINCH containers or in AS-1 in DEHP containers.

FIG. 11 is a plan view of a typical RBC storage container for use asdescribed herein.

FIG. 12 is a side view of the container of FIG. 11.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention is based upon the development of a combination redblood cell additive solution and storage bag lacking DEHP plasticizer.The invention generally relates to compositions and methods associatedwith the storage of red blood cells (RBC). In particular, it relates tothe combination of aqueous compositions for the storage of red bloodcells in non-DEHP blood storage bags and related methods for the storageof red blood cells. In some embodiments, the red blood cells that havebeen separated from whole blood collected in citrate phosphate dextrose(CPD) solution, its variant, citrate phosphate double dextrose (CP2D)solution, or by aphaeresis (removal of whole blood from a patient ordonor) in acid citrate dextrose (ACD formula A with 7.3 g/L citric acid,24.5 g/L dextrose monohydrate, and 22 g/L sodium citrate dihydrate orACD formula B with 4.4 g/L citric acid, 14.7 g/L dextrose monohydrate,and 13.2 g/L sodium citrate dihydrate) or similar solutions.

The published patents, patent applications, websites, company names, andscientific literature referred to herein establish the knowledge that isavailable to those with skill in the art and are hereby incorporated byreference in their entirety to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.Any conflict between any reference cited herein and the specificteachings of this specification shall be resolved in favor of thelatter.

DEFINITIONS

As used in this description and the accompanying claims, the followingterms shall have the meanings indicated, unless the context otherwiserequires:

For purposes of this disclosure, the term “in vivo recovery” is usedherein to indicate the fraction of stored RBCs that remains incirculation for 24 hours, after re-infusion into the original humandonor.

The term “non-DEHP” means a plasticizer that is not di-ethyhexylphthalate (DEHP) but is a plasticizer other than di-ethyhexyl phthalate(DEHP). In some embodiments, a non-DEHP plasticizer is a non-phthalateplasticizer (i.e., is not a phthalate plasticizer). In some embodiments,the non-DEHP plasticizer is not di(2-ethylhexyl) phthalate (DEHP), isnot the diisodecyl phthalate (DIDP), is not diisononyl phthalate (DINP),and/or is not di-(2-propyl heptyl) Phthlate (Palitinol 10-P). Anon-limiting non-DEHP plasticizer may be (but is not limited to) anyone, or combination of the following: butyl-n-trihexyl-citrate (BTHC);trimellitates; citrates such as citrate ester acetyltri-nbutyl citrate(ATBC); di, (2, ethyl, hexyl) terephthalate (DENT) (also known asDioctyl terephthalate (bis(2-ethylhexyl)benzene-1,4-dicarboxylate, orDOTP); tri, (2-ethyl hexyl)trimellitate (TEHTM); and diisononylcyclohexane-1,2-dicarboxylate (Hexamoll®/DINCH®, both registeredtrademarks owned by BASF). Note that another name for DINCH is1,2-cyclohexane dicarboxylic acid diisononyl ester.

The term “regulatory requirements” or “regulatory standards” means thecurrent requirements for approval for aqueous compositions for storageof red blood cells in a given jurisdiction. In the United States, theregulatory requirements are described in Title 21 of the U.S. Code ofFederal Regulations (e.g., Sections 606, 630, 640, 660, etc.) and in theregulations set forth by the U.S. Food and Drug Administration FDA/CBERsummary basis of approvals for RBC additive solutions, an example ofwhich is hereby incorporated by reference. In Europe, the equivalent isdescribed in the document entitled, “Guide to the preparation, use andquality assurance of blood components” by the Council of Europe. All ofthese regulations are hereby incorporated by reference. Other countriesand communities (e.g., Canada and Japan) have equivalent regulations.One measure of performance of an aqueous solution for the storage of redblood cells is hemolysis, where the RBCs at the end of their storagelife should have <1% hemolysis (95% confidence that at least 95% of thepopulation estimate will be less than 1%) in the U.S., and 90% of unitsshould be <0.8% hemolysis in EU member states. Another measure ofperformance is the in vivo recovery rates. The standard in the U.S. isthat mean 24 hour, post transfusion, in vivo red cell recovery at end ofstorage of at least 75% with standard deviation of at most 9%, and thelower limit of a one-sided 95% confidence interval for the populationproportion of successes is 70% or greater, and in Europe is achieving amean 24 hours post-transfusion in vivo recovery of no less than 75% ofthe transfused red blood cells.

The term “standard blood banking procedure” means the currently usedmethods for the preparation of packed RBCs and subsequent storage. Suchmethods are well known by those skilled in the art and are described inTitle 21 of the U.S. Code of Federal Regulations, in the guidanceentitled “Changes to an Approved Application: Biological Products—HumanBlood and Blood Components Intended for Transfusion of for FurtherManufacture,” dated July 2001, and the new draft guidelines of the sametitle announced by the U.S. Food and Drug Administration on May 31,2013; and in the “Technical Manual” and “Circular of Information for theUse of Human Blood and Blood Components” by the American Association ofBlood Banks and Instruction for Use and by manufacturers of bloodcollection systems, which are hereby incorporated by reference to theextent permitted by law. Specifically, standard blood banking proceduredoes not traditionally involve periodic mixing of the RBCs units duringstorage, mainly due to the time and expense of ensuring the mixing isdone in a proper and timely manner.

As used herein, “chloride” refers to anionic chloride. Thus, the term“chloride” includes anionic chloride and the salt forms thereof, such asmay be formed from chloride anion(s) and physiologically-acceptablecation(s). The term “chloride” is not intended to include compoundswherein the chloride atom is covalently bonded to, for example, a carbonatom covalently bonded to a chloride atom in an organic molecule.

As used herein, the phrase “physiologically-acceptable buffering agent”refers to buffering agents which yield cations and anions eithernormally found in the blood, plasma, or serum of a human, or that may betolerated when introduced into a human. Suitable cations includeprotons, ammonium cations and metal cations. Suitable metal cationsinclude, but are not limited to, the cationic forms of sodium,potassium, calcium, and magnesium, where sodium and potassium arepreferred, and sodium is more preferred. An ammonium cation, i.e., acompound of the formula R4N⁺ where R is hydrogen or an organic group,may be used so long as it is physiologically acceptable. In a preferredembodiment, the cation is selected from hydrogen (i.e., proton), sodium,potassium, calcium, magnesium, and combinations thereof. As used herein,“buffering agent” refers to an agent that adjusts and regulates the pHof a composition.

The additive solutions or compositions for the storage of RBCs describedherein are aqueous, that is, they are formulated in water. A preferredwater of the invention is treated in order that it is essentiallypyrogen-free and sterile.

As used herein, “mEq/L” refers to the concentration of a particularcomponent (solute) present in proportion to the amount of water present.More specifically, mEq/L refers to the number of milli-equivalents ofsolute per liter of water. Milli-equivalents per liter are calculated bymultiplying the moles per liter of solute by the number of chargedspecies (groups) per molecule of solute, which is then multiplied by afactor of 1,000.

One embodiment of the present invention provides a non-DEHP storage bagand an aqueous composition for storage of red blood cells at about 1 toabout 6.degree. C. The composition consists essentially of: adenine;dextrose; at least one non-metabolizable membrane-protectant sugar; anda pH buffering system. The pH buffering system comprises a combinationof physiologically acceptable buffering agents and must include at leastone agent that provides bicarbonate anions, at least one agent thatprovides phosphate anions, and at least one agent that provides sodiumcations. The invention contemplates that a single buffering salt maysatisfy more than one of these requirements. In some embodiments, thecomposition does not contain NaCl. In some embodiments, the compositiondoes not contain chloride.

Storage solutions for red blood cells have been developed before.Initially, storage compositions were designed to be acidic to preventthe caramelization of the glucose during the heat sterilizationperformed in the final production step. In the 1950s, adenine wasdiscovered to be useful as an additive and replaces the adenine lost bydeamination. In the 1970s it became desirable to remove the plasma fromthe collected whole blood for platelets and for the manufacture ofplasma derivatives. This, however, led to a reduction in the percent invivo RBC recovery/survival of the resulting packed RBC.

By “packed red blood cells” or “packed RBC” is simply meant aconcentration of the red blood cell component of whole blood, whetherthat whole blood is in a donated whole blood unit, or whether that wholeblood is circulating in a donor. For example, from a unit of wholeblood, packed RBC is simply the red blood cells in the whole bloodconcentrated in percentage while removing other non-RBC components ofthe whole blood (e.g., platelets, white blood cells, plasma, etc.). Insome embodiments, the packed red blood cells may be in the presence ofan anticoagulant (e.g., CPD, CP2D, or ACD). The packed RBC componentdoes not require the inclusion of every red blood cell in the wholeblood, and it will be understood that to remove non-RBC components fromthe whole blood (e.g., by filtration or centrifugation), some red bloodcells may be removed as well. However, the resulting packed red bloodcells will have a higher concentration of red blood cells as compared toother whole blood components (e.g., in a smaller volume). Any method toconcentrate red blood cells can be used to obtain packed red blood cellsincluding, without limitation, centrifugation, filtration (e.g.,filtration to reduce the white blood cell counts), or any other form ofseparation (e.g., erythrocytapheresis). It is to this packed RBCs thatthe additive solution is added, forming a suspension of red blood cellsin additive solution.

To circumvent this, compositions known in the art as additive solutions(AS) were developed to restore volume, nutrients, and other useful RBCstabilizers. Additive solution compositions for the preservation of redblood cells (RBCs) after their separation from whole blood are intendedto be tailored specifically to the needs of RBCs. The development ofcertain additive solutions extended RBC storage to 6 weeks in 1981. Redblood cells (RBCs) stored in these solutions, however, undergo steadydeterioration after about 6 weeks as determined by the inability of 75%of such cells to survive in the circulation for 24 hours afterre-infusion back into the human donor. It has been observed that duringcontinued refrigerated storage, glucose is consumed at a decreasingrate, as the concentration of metabolic waste, i.e. lactic acid andhydrogen ions, increases. Such a decrease in the rate of glucosemetabolism leads to depletion of adenosine triphosphate (ATP), whichdirectly correlates to the recovery of RBCs when the cells are returnedto the circulation. Additive solutions such as Adsol RTM (AS-1),Nutricel RTM (AS-3), Optisol RTM (AS-5), and ErythroSol RIM weredesigned to extend the storage of RBCs at 1-6° C. All three AdditiveSolutions (ASs) currently licensed in the U.S., AS-1, AS-3, and AS-5,contain saline, adenine, glucose and some citrate and/or mannitol as“membrane protectants.” AS-3 also contains monosodium phosphate.

Tables 1 and 2 shows the formulas of various additive solutions that canbe used in the combination products and methods described herein.

TABLE 1 Compositions of Additive Solution Compositions (components inmM) EAS-81/AS- AS-3 EAS-61 EAS-64 EAS-76v6 7/SOLX ® NaCl 70 26 75 30 —NaHCO3 — — 30 26 NaH2PO4 23 — — — Na2HPO4 — 12 9  9 12 Adenine  2  2 2 2  2 Na3Citrate 18 — — — Dextrose 55 110  50 50 80 Mannitol — 55 20 3055

TABLE 2 Additives (mM) AS-1 AS-3 AS-5 PAGGSM SOLX ® Dextrose 111 55.5 4552 80 Guanosine 1.5 Na3Citrate 20 NaH2PO4 23 Na2HPO4 32 12 Adenine 2 2.22.2 2 2 NaCl 154 70.1 150 72 Mannitol 41 45.4 55 55 Citric Acid 12NaHCO3 26In some embodiments, the pH of AS-3, prior to addition of the red bloodcells, is 5.8. In some embodiments, the pH of EAS-61, prior to additionof the red blood cells, is 8.3. In some embodiments, the pH of EAS-76v6,prior to addition of the red blood cells, is 8.4. In some embodiments,the pH of EAS-81, prior to addition of the red blood cells, is 8.5.

Each of the U.S.-licensed ASs meet the licensure requirements for 6-weekRBC storage, but fail to achieve 7-week storage. Presently licensed RBCadditive solution compositions were developed before the RBC storagelesion (defined herein as the sum of the survival- and/orfunction-limiting effects of storage on RBCs) was understood to be anapoptotic process.

Almost all of the whole blood collected now is made into components, andthe RBC fraction is stored as packed RBCs. For blood drawn into theadditive solution systems, Whole blood is drawn into anticoagulant (e.g.CPD, CP2D), RBCs are packed by centrifugation, plasma is removed so thatRBCs make up approximately 80% of the volume, and then 100 ml or 110 mLof additive solution is added sterilely for 450 mL or 500 mL whole bloodcollection, respectively. The resulting suspensions have a RBC volumefraction of approximately 55%. RBCs stored in the conventionalFDA-approved additive solutions can be stored for only 6 weeks with anacceptable 24-hour in vivo recovery,

To increase the time of acceptable in vivo recovery of RBCs re-infusedinto patients after a storage period, attempts have been made to improvethe additive solutions and storage processes. In “Studies In Red BloodCell Preservation-7. In vivo and in vitro Studies With A ModifiedPhosphate-Ammonium Additive Solution,” by Greenwalt et al., Vox. Sang.65:87-94 (1993), the authors determined that an experimental additivesolution (designated EAS-2) containing (in mM): 20 NH4Cl, 30 Na₂HPO₄, 2adenine, 110 dextrose, 55 mannitol, formulated at a pH of 7.15, isuseful in extending the storage shelf-life of human RBCs from thecurrent standard of 5-6 weeks to an improved standard of 8-9 weeks.

In “Studies in Red Blood Cell Preservation-8; Liquid Storage of RedCells in Glycerol-Containing Additive Solution,” Vox. Sang. 67:139-143(1994), Greenwalt et al. described an additive solution (designatedEAS-25) that allowed 73 percent in vivo recovery of packed red cellsafter nine weeks.

In “Extending the Storage of Red Cells at 4.degree. C.,” Transfus. Sci.15:105-115 (1994) by Meryman et al., acceptable viability of RBCs storedin very dilute suspensions at low hematocrit for as long as 27 weekswere demonstrated.

With respect to approved and commercially available products, theadditive solutions presently licensed in the U.S. work for only about 6weeks with an average recovery of about 80%. Two additive solutionspresently licensed in Europe work for about 7 weeks with an average invivo recoveries of 77% (ErythroSol from Baxter Healthcare, La Chatre,France) and 75% (PAGGS mannitol from Maco Pharma). Novel solutionsrecently described by Kurup et al. (Vox Sang 2003: 85:253-261) may beexpected to have shorter storage times because of the lower ATPconcentrations.

In response to the deficiencies in these prior findings, Hess andGreenwalt developed lower volume disodium phosphate-containing alkalineexperimental additive solutions (EASs) that partially neutralize theeffect of collecting blood into acidic anticoagulant solutions such asCPD (citrate-phosphate-dextrose) or CP2D(citrate-phosphate-double-dextrose), and showed that these EASs improvedRBC ATP concentrations, reduced hemolysis, and appeared to decrease RBCmembrane morphological changes and loss (see U.S. Pat. Nos. 6,150,085and 6,447,987 to Hess and Greenwalt, the complete disclosures of whichare fully incorporated herein by reference). Various EASs were shown tosupport between 9 and 12 weeks of storage. Although these EASs yieldedsuperior performance results, they contained sodium chloride and wereformulated to require a relatively large volume resulting in greaterdilution of the stored RBC, thus increasing the risk of hemodilution inmultiply transfused patient recipients. In addition, the presence ofsodium chloride created a solubility limit on the amount of bufferingsalts and phosphates that the system could sustain at desirable volumes.

Increased duration of RBC storage remains an important considerationduring periods when demand is high but intermittent, such as duringwartime, and for geographical regions that require transfusable bloodbut only on an inconsistent and sporadic basis. In fact, given thecurrent level of reported waste due to expiration of the safe storageperiod prior to realization of a demand in general, increasing theduration of time that RBCs may be safely stored is an ongoing ubiquitousconcern.

Thus, there is a need for RBC storage containers and RBC storagecompositions formulated to retain and/or enhance recovery andperformance benefits in the lower volumes of conventional additivesolutions. There is a continuing need in the blood storage andtransfusion art for improved RBC storage that results in longer storageduration, better in vivo recovery percentage, and improved physiologicalfunctioning of the transfused RBC. Consequently, there remains a needfor improved RBC storage compositions and processes of manufacturethereof. There is also a continuing need for additive compositions whichallow the RBC suspension to which the composition is added to bedirectly infused into humans, and which permit an acceptablepost-infusion recoverability of viable RBCs possessing enhancedphysiological functioning capabilities and lower rates of clearance fromthe infused patient's circulation.

A non-limiting aqueous composition for the storage of red blood cells isdescribed in U.S. Patent Pub. No. U.S. 2005-0233302 A1 by Hess andGreenwalt, which is hereby incorporated by reference in its entirety.One embodiment of the invention described in the Hess and Greenwaltapplication was an aqueous composition for the storage of red bloodcells wherein the aqueous composition consists essentially of adenine inan amount of about 1-3 mM, dextrose in an amount of from about 20 toabout 115 mM, unmetabolizable membrane-protectant sugar in an amount ofabout 15 to about 60 mM, sodium bicarbonate in an amount from about 20to about 130 mM, and disodium phosphate in an amount of from about 4 toabout 20 mM. This particular embodiment allowed better in vivo recoveryrates of RBCs stored for conventional 6 week storage periods, but alsoallowed for acceptable in vivo recovery rates of RBCs stored for longerperiods. Thus, a superior storage composition had been formulated.

Examples of widely used and accepted storage media are Adsol RTM andSAG-M, both available from Fenwal, Inc., of Lake Zurich, Ill.

Other additive solutions are disclosed in U.S. Patent ApplicationPublication Nos. 2009/0239208 and 2011/0117647 both of which areincorporated by reference herein in their entireties.

In one embodiment, the non-DEHP plasticizer is DINCH. In one embodiment,the composition consists essentially of adenine in an amount of about1-3 mM, dextrose in an amount of from about 20 to about 115 mM,nonmetabolizable membrane-protectant sugar in an amount of about 15 toabout 60 mM, sodium bicarbonate in an amount from about 20 to about 1 30mM, and disodium phosphate in an amount of from about 4 to about 20 mM.In another embodiment the composition consists essentially of adenine inan amount of about 2 mM, dextrose in an amount of from about 60 to about100 mM, nonnnetabolizable membraneprotectant sugar in an amount of about40 to about 60 mM, sodium bicarbonate in an amount of from about 22 toabout 40 mM, and disodium phosphate in an amount of from about 7 toabout 15 mM, and in a still further embodiment the composition consistsessentially of adenine in an amount of about 2 mM, dextrose in an amountof about 80 mM, nonmetabolizable membrane-protectant sugar in an amountof about 55 mM, sodium bicarbonate in an amount of about 26 mM, anddisodium phosphate in an amount of about 12 mM, and further wherein thecomposition has a pH of about 8.5.

In a still further embodiment, the at least one nonmetabolizablemembrane-protectant sugar is mannitol. In a still further embodiment,the at least one nonmetabolizable membrane-protectant sugar is a sugaralcohol, such as a monosaccharide-derived sugar alcohol. Some sugaralcohols, in particular the monosaccharide-derived sugar alcohols (e.g.,sorbitol, mannitol, xylitol, erythritol), are small hydrophilicmolecules that appear to diffuse readily through some lipid harriers andmay play an important role in cellular stability. Mannitol, inparticular, is a known antioxidant that acts as a hydroxyl radicalscavenger in vivo. It appears to play a substantial role in themaintenance of cell membrane integrity and is considered amembrane-protectant sugar. Other small polyols may also function asmembrane protectant sugars. It is significant to note that glucose andmannitol have the same mole weight, that is, 180 g/mole. Sugar alcoholsare not metabolized by the red blood cell.

Still further embodiments provide that the aqueous compositions have anosmolarity of from about 200 to about 340 mOsM. In some embodiments thecompositions have an osmolarity of from about 210 to about 310 mOsM. Insome embodiments the compositions have an osmolarity of from about 221to about 280 mOsM. In yet another embodiment the osmolarity is about 270mOsM.

As noted, RBCs metabolize glucose (d-glucose=“dextrose”) to make ATP.The waste products are lactate and protons. The protons accumulate,driving down the pH and inhibiting further metabolism. Bicarbonate hasbeen suggested as a buffer system wherein it combines with the protonsand, in the presence of RBC carbonic anhydrase, is converted to waterand carbon dioxide. In a storage container that permits diffusion of thecarbon dioxide, the reverse reaction is prevented and the reaction isdriven toward the formation of CO₂. A buffering system based onbicarbonate has considerable capacity. Bicarbonate in physiologicconcentrations in the additive solution creates the pCO₂ in the solutionthat drives the diffusion of up to 1 to 2 mmol of CO₂ from an about 100to about 800 mL capacity PVC bag (or from about 200 to about 700 mLcapacity, or from about 400 to about 600 mL capacity PVC bag) each week.However, previous attempts to formulate RBC storage additive solutionswith bicarbonate have failed with respect to increasing ATP synthesisand prolonging the effective storage period. For instance, Beutler(BAG-PM) described the addition of bicarbonate to RBC storage solutions,but failed to control for a high pH that led to rapid ATP depletion.

One preferred embodiment of the combination product provides that theaqueous compositions have a pH of from about 8 to about 10.5. In otherembodiments the pH is from about 8.2 to about 10. In other embodimentsthe pH is from about 8.2 to about 9.5. In further embodiments, the pH ofthe composition is from about 8.4 to about 8.6, and in still furtherembodiments the pH of the composition is about 8.5. Another embodimentis directed to the inventive compositions wherein the buffering systemhas a buffering capacity in the red blood cell (RBC) suspension to whichthe composition is added which increases by at least about 2 mEq betweena pH of 6.5 and 7.2 over a storage period of 6 weeks. The presentlydisclosed buffering system should provide a buffering capacity of atleast this value, but is capable of providing even greater bufferingcapacities to the RBC suspension thereby lengthening the storage periodeven further.

The present invention also provides method embodiments. In one suchembodiment a method of preserving red blood cells (RBCs) for a storageperiod is provided. The method comprises: (a) mixing a sample ofcollected whole blood containing the RBCs to be stored and plasma withan anticoagulant solution, thereby forming a suspension of collectedwhole blood; (b) treating the suspension of collected whole blood todeplete the plasma and concentrate the RBCs, thereby forming packed RBCs(either with or without reducing the leukocyte content of blood); (c)mixing the packed RBCs With an amount of an aqueous compositionsufficient to form a suspension of RBCs having about 35% to about 80%RBCs by volume; (d) cooling the suspension of RBCs to about 1 to about6° C.; and (e) storing the cooled suspension of RBCs according tostandard bank procedures known in the art. consists essentially ofadenine in an amount of about 1-3 mM, dextrose in an amount of fromabout 20 to about 115 mM, nonmetabolizable membrane-protectant sugar inan amount of about 15 to about 60 mM, sodium bicarbonate in an amountfrom about 20 to about 130 mM, and disodium phosphate in an amount offrom about 4 to about 20 mM. The solution may be divided in manufactureto separate components of the solution into different compartments(e.g., during sterilization). For example, the dextrose may be separatedfrom the phosphate, which may be separated from the sodium bicarbonate,which may be separated from the adenine, which may be separated from thenonmetabolizable membrane-protectant sugar. Of course, some componentsmay be combined (e.g., the nonmetabolizable membrane-protectant sugarand dextrose may be together and the bicarbonate and adenine may betogether.

Further provided are methods for storing packed RBCs in the compositionof one preferred embodiment in non-DEHP-PVC storage containers. Themethod of preserving red blood cells (RBCs) for a storage periodcomprises: (a) collecting a sample of whole blood containing the RBCs tobe stored and plasma in a blood storage container, wherein the bloodstorage container comprises PVC and a non-DEHP plasticizer, (b) mixingthe sample of collected whole blood with an anticoagulant solution,thereby forming a suspension of collected whole blood; (c) treating thesuspension of collected whole blood to deplete the plasma andconcentrate the RBCs, thereby forming packed RBCs (either with orwithout reducing the leukocyte content of blood); (d) mixing the packedRBCs with an amount of an aqueous composition sufficient to form asuspension of RBCs having about 35% to about 80% RBCs by volume; (e)cooling the suspension of RBCs to about 1 to about 6° C.; and (f)storing the cooled suspension of RBCs according to standard blood bankprocedures, wherein the aqueous composition consists essentially ofadenine in an amount of about 1-3 mM, dextrose in an amount of fromabout 20 to about 115 mM, unmetabolizable membrane-protectant sugar inan amount of about 15 to about 60 mM, sodium bicarbonate in an amountfrom about 20 to about 130 mM, and disodium phosphate in an amount offrom about 4 to about 20 mM.

Methods directed to the use of DINCH-PVC storage containers are alsoprovided. Methods according to the present invention directed tospecific ranges of the necessary ingredients of the composition are alsoprovided. In one method embodiment the composition consists essentiallyof adenine in an amount of about 1-3 mM, dextrose in an amount of fromabout 20 to about 115 mM, nonmetabolizable membrane-protectant sugar inan amount of about 15 to about 60 mM, sodium bicarbonate in an amountfrom about 20 to about 1 30 mM, and disodium phosphate in an amount offrom about 4 to about 20 mM. In another embodiment the compositionconsists essentially of adenine in an amount of about 2 mM, dextrose inan amount of from about 60 to about 100 mM, nonmetabolizablemembrane-protectant sugar in an amount of about 40 to about 60 mM,sodium bicarbonate in an amount of from about 22 to about 40 mM, anddisodium phosphate in an amount of from about 7 to about 15 mM, and in afurther embodiment the composition consists essentially of adenine in anamount of about 2 mM, dextrose in an amount of about 80 mM,nonmetabolizable membrane—protectant sugar in an amount of about 55 mM,sodium bicarbonate in an amount of about 26 mM, and disodium phosphatein an amount of about 12 mM, and further wherein the composition has apH of about 8.5.

It is well known in the red blood cell preservation arts that theconcentration of ATP in the red blood cell suspension system is the bestcorrelate of the health of the system. The red blood cell generates ATPthrough glycolysis via the glycolytic conversion of d-glucose (dextrose)ultimately to lactate. Hence, the concentration curve of lactate is agood indicator of ATP synthesis as well. Regardless of the preservationcapacity of the system, red blood cells have a finite life span and thecollected red blood cells include a normal distribution of red bloodcell ages and proximities to natural death. As no new RBCs are enteringthe preservation system, there is a limit to the maximum storage periodduration that will provide the requisite post-re-infusion in vivorecovery percentage. Hence, the ATP-generating capacity of the system asa whole will decrease over time, though; it is typical to see an initialincrease upon addition of an additive fluid as it provides nutrients inhigher than natural concentrations.

Without being bound by theory, it is believed that when stored in thecomposition component of the of the combination storage container andcomposition for storing red blood cells described herein, the increasedvolume of nutrient solution allows an increased mass of substrate to bedelivered at acceptable concentrations while providing solute fordilution of metabolic waste products thereby reducing feedbackinhibition of glucose metabolism. It is further postulated that anotherfeature of the additive solutions of the invention is that they produceswelling of the RBCs initially followed by a gradual reduction of redcell volume during storage. Such a process has been called “regulatedvolume decrease.” It is hypothesized that during this process either thetyrosine phosphatase activity present in the RBC is suppressed or thetyrosine kinase is activated. Both of these enzymes have beendemonstrated to be abundant in the membranes of these cells (Zipser, Y.and Kosower, N. S. (1996) Biochem. J. 314:881; Mallozzi C. et al. (1997)FASEB J. 11: 1281). It is anticipated that the net phosphorylation ofthe band 3 protein in the RBC membrane would result in the release ofphosphofructokinase, aldolase and glyceraldehyde-3-phosphatedehydrogenase in the cytoplasm from their bound state to band 3(Harrison, M. L. et al. (1991) J. Biol. Chem. 266:4106; Cossins, A. R.and Gibson J. S. (1997) J. Exper. Biol. 200:343; Low, P. S. et al.(1993) J. Biol. Chem. 268:14627; Low, P. S. et al. (1995) Protoplasma184:1961. The availability of these three enzymes in the glycolyticpathway would be expected to increase the metabolism of glucose by theRBC, thereby promoting the levels of ATP synthesis and ATP concentrationin the RBCs. So, the goal of formulating additive solution compositionsis to maintain the ATP synthesis at as high a rate as possible for aslong duration as possible.

A key to maximizing the ATP synthesis of the system is to keep the RBCintracellular pH at a level as close to 7.2 as possible without actuallyreaching it. During storage, the ATP concentration characteristicallyremains level or even increases for a period of time early in storageand then declines. When the RBC ATP concentration falls below 2.mu.mol/gHb, RBC recovery is typically below 75%. RBC's lose 2,3-DPG early instorage. The starting concentration is characteristically about 15umol/g Hb or about 1.1 mol/mol Hb. The concentration typically falls toone-tenth the starting amount in 7 to 10 days. The rate of synthesis of2,3-DPG is a function of pH, occurring in excess above pH 7.2 but withbreakdown favored below that pH. Attempts to increase 2,3-DPG synthesesby increasing storage-system pH have been limited by the mole for moleloss of ATP synthesis with each 2,3-DPG molecule formed. Thus, raisingRBC 2,3-DPG concentrations, something previously considered to bedesirable, actually tends to reduce RBC storage time.

A more acidic environment diminishes RBC metabolism. The pH of 7.2 isthe point wherein a mechanism, known as the Rappaport shunt (see Hess etal. “Alkaline CPD and the preservation of red blood cell 2,3-DPG” (2002)Transfusion, 42:747-752, fully incorporated herein by reference) istriggered whereby 2,3-DPG is synthesized from 1,3-DPG, consuming thephosphate needed for the synthesis of ATP and, additionally, routingaround a glycolytic step which produces two of the glycolyticallygenerated ATPs. The net effect to the system is a depletion of ATP. Ifthe intracellular pH can be maintained below 7.2, the shunt iseffectively closed down and ATP synthesis is maximized. In a naturalstate, the shunt operates to some extent and the production andmaintenance of some 2,3-DPG is important to other cellular events.However, for purposes of preservation of the red blood cell duringstorage outside of the in vivo environment, minimization of the shuntoperation is desirable.

Therefore, of the composition component of the combination storagecontainer and composition for storing red blood cells, the compositioncomprises a pH buffering system that is present in an amount sufficientfor the composition to be operable to maintain a pH of a red blood cell(RBC) suspension to which the composition is added at a value sufficientto establish and maintain during a storage period a reaction equilibriumin the red blood cell that favors glycolysis over synthesis of2,3-diphosphoglycerate (DPG) from 1,3-DPG, thereby generating a net gainin adenosine tri phosphate (ATP) synthesis with respect to the reactionequilibrium during the storage period. A specific embodiment providesthat the composition is operable to maintain the pH of the RBCsuspension to which the composition has been added at between about 6.4and 7.4. In more specific embodiments, the composition is operable tomaintain the pH of the red blood cell (RBC) suspension to which thecomposition has been added at between 7.0 and less than about 7.2. Invery specific embodiments, the composition is operable to maintain thepH of the red blood cell (RBC) suspension to which the composition hasbeen added at a value greater than about 7.1 and less than 7.2.

In further embodiments of the composition component of the combinationstorage container and composition for storing red blood cells, thecomposition is substantially free of chloride. This lack of chloridesurprisingly yields no negative effect on the system and permits theaddition of increased amounts of the buffering system to provideadditional pH buffering. In some embodiments, the aqueous compositionfor storage of red blood cells stores the RBCs at about 1 to about 6° C.This composition comprises: adenine; dextrose; at least onenonmetabolizable membrane-protectant sugar; and a pH buffering system.The pH buffering system comprises a combination of physiologicallyacceptable buffering agents including at least one agent providingbicarbonate anions, at least one agent providing phosphate anions, andat least one agent providing sodium cations. In some embodiments, thecomposition (prior to the addition of the red blood cells) does notcontain chloride. In some embodiments, the composition (prior to theaddition of the red blood cells) does not contain citrate. The pHbuffering system is present in an amount sufficient for the compositionto be operable to maintain a pH of a red blood cell (RBC) suspension towhich the composition is added at a value sufficient to establish andmaintain during a storage period a reaction equilibrium in the red bloodcell that favors glycolysis over synthesis of 2,3-diphosphoglycerate(DPG) from 1,3-DPG, thereby generating a net gain in adenosine triphosphate (ATP) with respect to the reaction equilibrium during thestorage period. The composition is substantially free of exogenouslyderived chloride ions. As used herein, “substantially free ofexogenously derived chloride ions” is defined as whatever theconcentration of chloride ions is given that no source of chloride ionshas been added to the composition.

Additional embodiments are directed to a suspension of red blood cellscomprising any of the compositions within the blood storage bag, andembodiments wherein the suspension is suitable for direct infusion intoa patient in need of such an infusion.

In further embodiments of the composition component of the combinationstorage container and composition for storing red blood cells, at leastone agent providing sodium cations in the composition is selected fromthe group consisting of sodium bicarbonate, disodium phosphate,monosodium phosphate and combinations thereof. In a more specificembodiment the at least one agent providing bicarbonate anions is sodiumbicarbonate. Additional embodiments provide that the at least one agentproviding phosphate ions is selected from the group consisting of sodiumphosphate, disodium phosphate, trisodium phosphate, and combinationsthereof. In more specific embodiments the at least one agent providingphosphate ions is disodium phosphate. In other embodiments of theinventive composition the combination of physiologically acceptablebuffering agents additionally comprises at least one agent providing aphysiologically acceptable cation selected from the group consisting ofH⁺, potassium, ammonium, magnesium and combinations thereof. In someembodiments, the at least one agent providing sodium cations in thecomposition is not sodium chloride. In some embodiments, the at leastone agent providing sodium cations in the composition is not sodiumcitrate.

In a further embodiment of the composition component of the combinationstorage container and composition for storing red blood cells, at leastone non-metabolizable membrane-protectant sugar is mannitol. In someembodiments, the at least one non-metabolizable membrane-protectantsugar is a sugar alcohol (e.g., a monosaccharide-derived sugar alcohol).Some sugar alcohols, in particular the monosaccharide-derived sugaralcohols (e.g., sorbitol, mannitol, xylitol, erythritol), are smallhydrophilic molecules that appear to diffuse readily through some lipidbarriers and may play an important role in cellular stability. Mannitol,in particular, is a known antioxidant that acts as a hydroxyl radicalscavenger in vivo. It appears to play a substantial role in themaintenance of cell membrane integrity and is considered amembrane-protectant sugar. Other small polyols may also function asmembrane protectant sugars. It is significant to note that glucose andmannitol have the same mole weight, that is, approximately 180 g/mole.Sugar alcohols are not metabolized by the red blood cell.

As used herein, the reported osmolarity is an empirically derived value.Osmolarity is a measure of the osmotic pressure exerted by a solutionacross a perfect semi-permeable membrane (one which allows free passageof water and completely prevents movement of solute) compared to purewater. Osmolarity is dependent on the number of particles in solutionbut independent of the nature of the particles. The osmolarity of asimple solution is equal to the molarity times the number of particlesper molecule. Real solutions may be much more complex. Proteins withmany equivalents/L may only contribute a small amount to the osmolarity,since they consist of a few very large “particles”. Not all ions arefree in a solution. Cations may be bound to other anions or to proteins.Not all the solution volume is aqueous. To be truly accurate, all thesefactors should be included in the calculation.

Tonicity, a value highly related to osmolarity and somewhat more usefulfor describing biocellular conditions, is a measure of the osmoticpressure that a substance can exert across a cell membrane, compared toblood plasma. Osmolarity measures the effective gradient for waterassuming that all the osmotic solute is completely impermeant. It issimply a count of the number of dissolved particles. A 300 mM solutionof glucose and a 150 mM solution of NaCl each have the same osmolarity,for example. However, a cell placed in each of these solutions wouldbehave very differently. Tonicity is a functional term describing thetendency of a solution to resist expansion of the intracellular volume.

In additional embodiments, the composition component of the combinationstorage container and composition for storing red blood cells, has anosmolarity of from about 200 to about 340 mOsmol/liter prior to theaddition of the red blood cells. In some embodiments the compositionshave an osmolarity of from about 210 to about 310 mOsM. In more specificembodiments the compositions have an osmolarity of from about 221 toabout 280 mOsmol/liter. In another specific embodiment, the osmolarityis about 244 mOsM (i.e., mOsmol/liter). In another specific embodiment,the osmolarity is about 225 mOsM.

As noted above, RBCs metabolize glucose (d-glucose=“dextrose”) to makeATP. The waste products are lactate and protons. The protons accumulate,driving down the pH and inhibiting further metabolism. Bicarbonate hasbeen suggested as a buffer system wherein it combines with the protonsand, in the presence of RBC carbonic anhydrase, is converted to waterand carbon dioxide. In a storage container that permits diffusion of thecarbon dioxide, the reverse reaction is prevented and the reaction isdriven toward the formation of CO2. A buffering system based onbicarbonate has considerable capacity. Bicarbonate in physiologicconcentrations in the additive solution creates the pCO₂ in the solutionthat drives the diffusion of up to 1 to 2 mmol of CO₂ from an about anabout 100 to about 800 mL capacity PVC bag (or from about 200 to about700 mL capacity, or from about 400 to about 600 mL capacity PVC bag)each week. However, previous attempts to formulate RBC storage additivesolutions with bicarbonate have failed with respect to increasing ATPsynthesis and prolonging the effective storage period. For instance,Beutler (BAG-PM) described the addition of bicarbonate to RBC storagesolutions, but failed to control for a high pH that led to rapid ATPdepletion.

In discovering that saline (i.e., NaCl) is not a necessary ingredient toRBC additive solution compositions, and that the concentration ofdextrose could be lowered without negative effects on ATP synthesis, theresultant increased “play” in solution parameters allows the fine-tuningof the pH buffering system. The presently disclosed buffering systemprovides not only an initially appropriate pH to the additive solutioncomposition, but is able to impart to the RBC suspension a pH that, inturn, modulates the intracellular pH of the RBC to maximize ATPsynthesis. The buffering system achieves these pH modulation targetsover the storage period. Hence, the buffering capacity or strength ofthe pH buffering system is deliberately controlled. One embodiment ofthe present inventive compositions provide that the composition have apH of from about 8 to about 10.5. In more specific embodiments the pH isfrom about 8.2 to about 10. In even more specific embodiments the pH ofthe composition is from about 8.4 to about 9.5. In even more specificembodiments the pH of the composition is from about 8.4 to about 9, andin a very specific embodiment the pH of the composition is about 8.5.

Another embodiment is directed to the inventive compositions wherein thebuffering system has a buffering capacity in the red blood cell (RBC)suspension to which the composition is added which increases by at leastabout 2 mEq between a pH of 6.5 and 7.2 over a storage period of 6weeks. The presently disclosed buffering system should provide abuffering capacity of at least this value, but is capable of providingeven greater buffering capacities to the RBC suspension therebylengthening the storage period even further.

Ranges for the necessary composition ingredients that permit theinstantly disclosed advantages are provided. In one embodiment of theinventive composition, the composition comprises adenine in an amount ofabout 1-3 mM, dextrose in an amount of from about 20 to about 115 mM,un-metabolizable membrane-protectant sugar in an amount of about 15 toabout 60 mM, sodium bicarbonate in an amount from about 20 to about 130mM, and disodium phosphate in an amount of from about 4 to about 20 mM.In a more specific embodiment the composition comprises adenine in anamount of about 2 mM, dextrose in an amount of from about 60 to about100 mM, unmetabolizable membrane-protectant sugar in an amount of about40 to about 60 mM, sodium bicarbonate in an amount of from about 22 toabout 40 mM, and disodium phosphate in an amount of from about 7 toabout 15 mM. In an even more specific embodiment the compositioncomprises adenine in an amount of about 2 mM, dextrose in an amount ofabout 80 mM, unmetabolizable membrane-protectant sugar in an amount ofabout 55 mM, sodium bicarbonate in an amount of about 26 mM, anddisodium phosphate in an amount of about 12 mM. In some embodiments, thecomposition has a pH of about 8.5.

In another embodiment, the present invention also provides methodembodiments. In one such embodiment a method of preserving red bloodcells (RBCs) for a storage period is provided. The method comprises: (a)mixing a sample of collected whole blood containing the RBCs to bestored and plasma with an anticoagulant solution, thereby forming asuspension of collected whole blood; (b) treating the suspension ofcollected whole blood to deplete the plasma and concentrate the RBCs,thereby forming packed RBCs (either with or without reducing theleukocyte content of blood); (c) mixing the packed RBCs with an amountof an aqueous composition sufficient to form a suspension of RBCs havingabout 35% to about 80% RBCs by volume; (d) cooling the suspension ofRBCs to about 1 to about 6° C.; and (e) storing the cooled suspension ofRBCs in a non-DEHP container according to standard bank procedures knownin the art. The aqueous composition consists essentially of: adenine;dextrose; at least one non-metabolizable membrane-protectant sugar; anda pH buffering system. The pH buffering system comprises a combinationof physiologically acceptable buffering agents including at least oneagent providing bicarbonate anions, at least one agent providingphosphate anions, and at least one agent providing sodium cations,wherein the pH buffering system is present in an amount sufficient forthe composition to be operable to maintain a pH of a red blood cell(RBC) suspension to which the composition is added at a value sufficientto establish and maintain during a storage period a reaction equilibriumin the red blood cell that favors glycolysis over synthesis of2,3-diphosphoglycerate (DPG) from 1,3-DPG, thereby generating a net gainin adenosine tri phosphate (ATP) synthesis with respect to the reactionequilibrium during the storage period. The solution is divided inmanufacture to separate the dextrose and the phosphate and bicarbonateduring heat sterilization.

RBCs useful in the present invention are those that have been separatedfrom their plasma and resuspended in an anticoagulant solution in thenormal course of component manufacture. Briefly stated, a standard wholeblood sample (450+ 45 ml) containing RBCs and plasma is mixed with ananticoagulation solution (about 63 ml) to form a suspension of wholeblood. Proportional increases or decreases in solution volumes toreflect different donor blood volumes such as 400+ 40 ml-500+ 50 ml canalso be used. The whole blood suspension is thereafter centrifuged toseparate the RBCs from the blood plasma thereby forming packed RBCs. Theperformance of the overall process is improved by leukocyte reductionusing conventional techniques.

Suitable anticoagulants include conventional anticoagulants known forstorage of RBCs. Preferably; the anticoagulants include citrateanticoagulants having a pH of 5.5 to 8.0, e.g. CPD, half-strength CPDand the like. The most preferred anticoagulant is CPD or CP2D.

The RBC suspension is then stored in a storage back lacking DEHP, forexample, a polyvinyl chloride (PVC) blood storage bags lacking DEHPusing either the collection bag or PVC transfer packs of different sizesdepending on the volume of the stored aliquot. The RBC suspension isstored at about 1 to 6° C. according to standard blood bank procedure asdescribed in Clinical-Practice of Blood Transfusion editors: Petz &Swisher, Churchill-Livingston publishers, N.Y., 1981. All documentscited herein infra and supra are hereby incorporated by referencethereto.

In a specific embodiment, the suspension of RBCs is suitable for directinfusion into a patient in need of such an infusion. While PVC bloodstorage bags are the industry-approved standard; the present inventioncontemplates storage in a wide variety of bags adapted for RBCsuspension storage, for example, by including appropriate plastisizersas needed. Ingredients related to the bag or container component of RBCstorage technology are not discussed herein but it will be readilyapparent to one of ordinary skill in the art that many containertechnologies may be employed to practice the present invention.

The composition component of the combination storage bag and compositionproduct described herein can also be used to rehydrate lyophilized RBC(e.g., in the thawing of stored frozen blood or blood component (e.g.,RBC)), or pathogen inactivated blood or blood component (e.g., RBC).

In specific embodiments of the inventive method of preserving RBCs, thecomposition contains at least one non-metabolizable membrane-protectantsugar that is a monosaccharide derived sugar alcohol and in a morespecific embodiment the non-metabolizable membrane-protectant sugar ismannitol. In additional embodiments of the method, the at least oneagent providing sodium cations is selected from the group consisting ofsodium bicarbonate, disodium phosphate, and combinations thereof. Inspecific embodiments the at least one agent providing bicarbonate anionsis sodium bicarbonate. Further embodiments are directed to the inventivemethod of preserving RBCs wherein the at least one agent providingphosphate ions is selected from the group consisting of sodiumphosphate, disodium phosphate, trisodium phosphate, and combinationsthereof, and in more specific embodiments that at least one agentproviding phosphate ions is disodium phosphate. In other embodiments ofthe inventive method the combination of physiologically acceptablebuffering agents additionally comprises at least one agent providing aphysiologically acceptable cation selected from the group consisting ofH+, potassium, ammonium, magnesium and combinations thereof.

The present invention also provides embodiments preserving RBCs in thecombination non-DEHP storage bag and composition wherein thecomposition, prior to addition of the RBCs, has an osmolarity of fromabout 200 to about 340 mOsm. In some embodiments the compositions havean osmolarity of from about 210 to about 310 mOsM. In furtherembodiments the osmolarity of the composition, prior to addition of theRBCs, is from about 221 to about 280 mOsm. In specific embodiments theosmolarity of the composition, prior to addition of the RBCs, is about225 mOsm. In other embodiments, the composition, prior to addition ofthe RBCs, has a pH of from about 8 to about 10.5. In specificembodiments the pH is from about 8.2 to about 10. and in more specificembodiments the pH of the composition, prior to addition of the RBCs, isfrom about 8.4 to about 9.6. In a very specific embodiment the pH of thecomposition, prior to addition of the RBCs, is about 8.5. An additionalembodiment of the combination non-DEHP storage bag and composition forpreserving RBCs described herein provides that the buffering system hasa buffering capacity in the red blood cell (RBC) suspension to which thecomposition is added which increases by 2 mEq between a pH of 6.5 and7.2 over a storage period of 6 weeks, or over a storage period of 8weeks, or a storage period of 10 weeks, or a storage period of 12 weeks.

The present invention also provides embodiments preserving RBCs in thecombination non-DEHP storage bag and composition wherein the compositionis operable to maintain the pH of the red blood cell (RBC) suspension towhich the composition has been added at between about 6.4 and about 7.4.In specific method embodiments the composition is operable to maintainthe pH of the red blood cell (RBC) suspension to which the compositionhas been added at between 7.0 and less than about 7.2, and in even morespecific method embodiments the composition is operable to maintain thepH of the red blood cell (RBC) suspension to which the composition hasbeen added at a value greater than about 7.1 and less than 7.2.

Methods according to the present invention directed to specific rangesof the necessary ingredients of the composition component of thecombination storage bag and composition are also provided. In one methodembodiment the composition comprises adenine in an amount of about 1-3mM, dextrose in an amount of from about 20 to about 115 mM,un-metabolizable membrane-protectant sugar in an amount of about 15 toabout 60 mM, sodium bicarbonate in an amount from about 20 to about 130mM, and disodium phosphate in an amount of from about 4 to about 20 mM.In a more specific embodiment the composition comprises adenine in anamount of about 2 mM, dextrose in an amount of from about 60 to about100 mM, unmetabolizable membrane-protectant sugar in an amount of about40 to about 60 mM, sodium bicarbonate in an amount of from about 22 toabout 40 mM, and disodium phosphate in an amount of from about 7 toabout 15 mM, and in a very specific embodiment the composition comprisesadenine in an amount of about 2 mM, dextrose in an amount of about 80mM, unmetabolizable membrane-protectant sugar in an amount of about 55mM, sodium bicarbonate in an amount of about 26 mM, and disodiumphosphate in an amount of about 12 mM, and further wherein thecomposition has a pH of about 8.5.

In accordance with the present disclosure, the composition (i.e., theadditive solution) component of the combination storage bag andcomposition is added to the packed RBC suspension in an amountsufficient to provide a therapeutically effective amount of in vivorecoverable RBCs in the cell suspension. Preferably, the additivesolution is added at a volume ranging from about 60 ml to about 400 ml,preferably about 100 to about 150 ml, most preferably about 110 ml. Thesolution is typically used in a 1:4.5 volume ratio of solution to wholeblood collected (100 mL for a 450 mL whole blood collection, 110 mL fora 500 mL whole blood collection, or equivalent). In specific embodimentsof the present inventive methods of preserving RBCs, the volume ratio ofthe composition to the collected whole blood is about 1:4.5. In a morespecific embodiment the volume of the composition is about 110 mL andthe volume of the collected whole blood is about 500 mL.

In further embodiments, the composition (i.e., the additive solution)component of the combination storage bag and composition comprises anaqueous solution of adenine, dextrose, Na2HPO4, mannitol, and at leastone physiologically acceptable sodium salt, in concentrations suitableto preserve RBCs. In general, the composition may contain adenine fromabout 1 to 3 mM, dextrose from about 20 to 115 mM, Na2HPO4 from about 4to 15 mM (where a combination of Na2HPO4 and NaH2PO4 can also be used),mannitol from about 15 to 60 mM, and at least one physiologicallyacceptable sodium salt from about 20 to 130 mM. Preferably, adenine isabout 2 mM, dextrose is about 50 to 110 mM, Na2HPO4 is about 9 to 12 mM,mannitol is about 20 to 50 mM, and at least one physiologicallyacceptable sodium salt is about 25 to 75 mM. Suitable sodium saltsuseful in the composition include those salt compounds containing asodium cation which are physiologically acceptable in humans.Non-limiting sodium salts include sodium chloride, sodium acetate,sodium citrate, sodium bicarbonate, and the like. In some embodiments,the sodium salt specifically excludes sodium chloride. In someembodiments, the sodium salt specifically excludes sodium citrate. Ofcourse, once the red blood cells are added to the composition, the redblood cells will add chloride ions (from plasma and the cellsthemselves) and citrate (from the CPD anti-coagulant). In someembodiments, the composition contains about 0 to 100 mM of sodiumchloride and/or 0 to 53 mM of sodium acetate. The pH of the composition(prior to RBC addition) is maintained in a range of about 7 to 10.5 atroom temperature. Preferably, the pH of the composition (prior to RBCaddition) is in the range of about 8 to 8.8. Most preferably, the pH ofthe composition is about 8.4 to about 8.6 prior to the addition of anyred blood cells.

The osmolarity of the composition (i.e., the additive solution)component of the combination storage bag and composition is in the rangeof about 200 to 340 mOsM prior to the addition of any red blood cells.In some embodiments the compositions have an osmolarity of from about210 to about 310 mOsM. Preferably, the osmolarity of the composition(i.e., the additive solution) component of the combination storage bagand composition is in the range of about 221 to 280 mOsM prior to theaddition of any red blood cells. Most preferably, the osmolarity of thecomposition (i.e., the additive solution) component of the combinationstorage bag and composition is about 221 to 256 mOsM prior to theaddition of any red blood cells.

The RBC volume fraction in the cell suspension, i.e. after addition ofcomposition (i.e., the additive solution) component of the combinationstorage bag and composition, is about 27 to 80% of the total suspension.More preferably, the RBC volume fraction in the cell suspension afteraddition of composition (i.e., the additive solution) component of thecombination storage bag and composition is about 35 to about 50%. Mostpreferably, the RBC volume fraction in the cell suspension afteraddition of composition (i.e., the additive solution) component of thecombination storage bag and composition is about 43% of the totalsuspension.

FIG. 1 shows an example of the state of the art. (“Exploratory In VitroStudy of Red Blood Cell Storage Container with an AlternativePlasticizer,” Larry Dumont et al. 2009, Oral and Poster Abstractpresentation (3149, Basic Science and Clinical Practice in BloodTransfusion Poster II, 51^(th) ASH Annual Meeting and Exposit^(o), NewOrleans, La.). RBCs were collected from healthy volunteer subjects intoCPD standard collection sets. The blood was combined into pools of twoABO identical RBCs, divided, leukocyte-reduced, centrifuged andseparated into plasma and packed RBC, then additive solution (AS-5) wasadded to the RBC, and the RBC/AS5 were transferred into 2 study bags(matched pair) for storage up to 42 days under standard blood bankconditions of 4° C. Part A shows in vitro characteristics of stored RBCsin DINCH bags that were mixed weekly (right of part A of FIG. 1),compared with RBCs stored in DINCH bags that were not mixed weekly (leftof part A of FIG. 1). Mixing the bags during storage clearly helps theperformance of the aqueous composition used in combination with DINCHbags. Part B shows stored RBCs in DEHP bags (without mixing; “Control”under part B of FIG. 1), compared with RBCs stored in DINCH bags (withmixing (“Dinch” under part B of FIG. 1). Each panel represents theresults of standard in vitro RBC characteristics determined after 42days of storage. The top panels show percent hemolysis. The middlepanels show extracellular potassium. The bottom panels show ATP. Bycomparing the results of the performance of aqueous storage compositionsin DINCH and DEHP bags of Part B, DINCH bags, with mixing, provided atleast equivalent protection against hemolysis and potassium leakage.

Additional data from Larry Dumont et al., supra is provided in Table3—Day 42 in vitro characterization (mean+/−SD, range; limit ofquantitation for DEHP is 0.2 ug/ml).

TABLE 3 Part A - DINCH Bag (n = 6 pools) Part B (n = 6 pools) No MixMixed p Control DEHP DINCH p Lactate (mM) 32.7 ± 3.0 35.1 ± 3.1 0.01131.1 ± 3.1 33.1 ± 2.8 0.0045 27.5-35.0 30.5-39.0   27-35.5 29.5-37.0Glucose (mg/dL) 242 ± 31 242 ± 34 0.905  274 ± 3 5 268 ± 37 0.0255215-296 208-298 227-321 221-319 Morphology  48 ± 15  58 ± 14 0.01 64 ± 6 62 ± 10 0.6724 RBC (0-100) 23-60 42-73 54-68 51-75 MCV (fL)  100 ± 2.998.7 ± 2.7 0.0002 101.6 ± 3.7  100.6 ± 3.7  0.0055  97-105  96-103  98-107.8  96.9-106.4 Supernatant ND ND — Median = 14.9 <0.2 — DEHP(μg/mL) <0.2-31.1These results showed that periodic mixing of RBC stored in DINCH bagsprovided additional protection against hemolysis over 42 days ofstorage. DINCH plasticizer provided at least equivalent protectionagainst hemolysis and potassium leakage for RBC stored in additivesolution for 42 days.

However, periodic mixing is not practical with current blood bankingmethodologies. The combination product and methods described hereinprovide means for storing red blood cells for at least 42 days withouthaving to periodically mix the stored red blood cells.

Accordingly, study was done comparing the hemolysis levels of red bloodcells stored either in SOLX® (also called EAS-81 and AS-7) additivesolution within either a DEHP bag, a DINCH bag, or a BTHC bag. To dothis, the pooled whole blood collected (plus CPD) was processed usingstandard blood bank procedures, either within eight hours aftercollection or after a room temperature hold of 24 hours. The resultingpacked RBCs were then combined with SOLX additive solution, split intovarious test groups and stored either in DEHP (black bars in FIG. 2),DINCH (medium gray bars in FIG. 2), or BTHC (light gray bars in FIG. 2)bags. Hemolysis was sampled weekly at weeks five through nine, ascompared to DEHP as the control (hence, the DEHP bars (black) are always1.0 in FIG. 2). ATP was measured at eight weeks. Morphology of the RBCswas scored at eleven weeks. As shown in FIG. 2, the mean hemolysis ratio(hemolysis standardized in each pool by DEHP unit) shows that RBCsstored in alternatively plasticized PVC containers result in an averageof 50% higher hemolysis than in DEHP plasticized containers. The higherhemolysis observed with alternative plasticizers are consistent withthose reported in the literature for RBCs stored in conventionaladditives.

Further experiments led to the surprising discovery that improvement inthe RBC integrity and storage capabilities observed with RBCs stored ina composition consisting essentially of adenine in an amount of about1-3 mM, dextrose in an amount of from about 20 to about 115 mM,unmetabolizable membrane-protectant sugar in an amount of about 15 toabout 60 mM, sodium bicarbonate in an amount from about 20 to about 130mM, and disodium phosphate in an amount of from about 4 to about 20 mMstored in a non-DEHP container as compared to AS-1 RBCs stored instandard DEHP containers will negate the lower RBC integrity and higherhemolysis observed with RBCs stored in alternative plasticized PVCcontainers.

A study that compared RBCs stored in DINCH containers lacking DEHP in acomposition consisting essentially of adenine in an amount of about 1-3mM, dextrose in an amount of from about 20 to about 115 mM,unmetabolizable membrane-protectant sugar in an amount of about 15 toabout 60 mM, sodium bicarbonate in an amount from about 20 to about 130mM, and disodium phosphate in an amount of from about 4 to about 20 mM,a preferred embodiment, to RBCs stored in DEHP containers in AS-1additive solution was next performed.

This paired study was carried out by collecting whole blood units intoCPD, as is standard, pooling ABO compatible units in pairs, and thensplitting them into individual blood units of equivalent volume. The tenpools were filtered and leukocyte reduced. The resulting RBCs werepacked and the plasma drawn off following standard procedures. Thepacked RBCs were then resuspended in either the composition of apreferred embodiment (in this case, SOLX) or AS-1, and stored in DINCHand DEHP containers, respectively.

Specifically, the following was done: Whole blood (WB) units (500±50 mL)were collected into Fenwal 500 mL collection sets with 70 mL CPDanticoagulant. Pairs of ABO/Rh compatible units were pooled and equallysplit between LEUKOSEP® HWB-600-XL Whole Blood Leukocyte ReductionFiltration System with SOLX®/DINCH® (Test) and a Blood-Pack Unit withIntegral RZ2000 WB Leukocyte Reduction Filter with AS-1/DEHP (FenwalInc., Lake Zurich, Ill.) (Control). Test and Control sets werepreviously drained of anticoagulant.

WB units were filtered and processed at room temperature (RT) withineight hours of collection. Test units were filtered using the LEUKOSEP®HWB-600-XL filter and Control units using the RZ-2000 filter. WB wascentrifuged, plasma removed and 110 mL SOLX® added to the Test RBC unitsand 110 mL AS-1 added to the Control RBC units.

RBC were stored for 42 days at 1-6° C. without periodic mixing. Testingwas performed on WB post-filtration and RBC on Days 0 and 42 of storage.Evaluations included residual WBC (BD FACS Calibur), CBC (SysmexXE-2100D), blood gases (Roche Cobas b 221), supernatant hemoglobin(HemoCue Plasma/Low Hb), ATP (Rolf Greiner), RBC morphology,microvesicle protein analysis, and DINCH® and DEHP levels.

Data was collected on ten pairs of WB units. All Test and Control unitshad a post-filtration rWBC content of <5×10⁶/unit. Results of in vitromeasurements are presented in Table 4.

TABLE 4 Day 0 (Post-Processing) Day 42 Test Control Test Control(SOLX/DINCH) (AS-1/DEHP) (SOLX/DINCH) (AS-1/DEHP) Hemolysis (%) 0.03 +/−0.02 0.02 +/− 0.00 0.36 ± 0.12  0.35 +/− 0.13 ATP 3.97 +/− 0.28 4.20 +/−0.34  3.55 ± 0.51*  3.40 +/− 0.42 (μmol/gHb) ATP (% of Day 0) NA NA 89.2± 9.8* 80.8 +/− 6.3 Potassium 0.79 +/− 0.14 0.71 +/− 0.18 45.4 ± 4.2*43.8 +/− 3.8 (mEq/L) RBC 98.9 +/− 0.6  97.0 +/− 2.3  67.5 ± 11.7  66.7+/− 12.0 Morphology (0-100) Microvesicle NA NA 21.5 ± 7.0* 24.9 +/− 4.8Protein (mg/100 mL RBC) DINCH (ppm) <1.0** NA 2.50 ± 0.86 NA DEHP (ppm)<1.0** <1.0** <1.0** 20.9 +/− 5.0 Mean +/− standard deviation N = 10*Statistically significant difference from Day 42 Control (p < 0.05)**Lower Limit of Quantitation = 1.0 ppm

From these studies, it was found that RBC stored in DINCH plasticizedPVC bags and SOLX additive demonstrated comparable quality measures ascompared to conventional licensed AS-1 stored in DEHP plasticizedcontainers. The results demonstrated the potential cold storage of SOLXRBC in DINCH/PVC containers for 42 days utilizing a standard additivevolume (110 mL per 500 mL WB collection) and without mixing, specializedequipment or processing.

During 42 days of storage, hemolysis was <0.59% for both Test andControl RBC. Potassium levels were statistically higher for Test RBCcompared to Control, but were not considered to be clinicallysignificant.

FIG. 3 shows the result of the morphology scoring at day 42 of storage.The composition of a preferred embodiment (SOLX, in this case) in DINCHbags shows almost identical results to AS-1 in DEHP bags. FIG. 4 showsthe amount of microvesicle protein after 43 days of storage in the twostudy groups. As microvesicles are an indicator of cell damage anddeath, the less protein measured, the better the cells' health isconsidered to be. The SOLX composition of a preferred embodiment inDINCH containers surprisingly showed a statistically significant(p<0.05) improvement over the conventional AS-1/DEHP combination. FIG. 5shows the amount of ATP (as a percentage of initial ATP levels) in thetwo study groups. ATP is a well-known corollary of RBC health. Again,the SOLX/DINCH combination showed a statistically significant (p<0.05)greater amount of ATP at the end of 42 days than found for the AS-1/DEHPcombination. Further, and potentially even more surprising, hemolysis(as measured by percent of hemoglobin content), as shown in FIGS. 6A and6B (FIG. 6A shows the mean of the ten pools, FIG. 6B shows theindividual pools) was almost identical between the two groups.

The results in FIGS. 3-6B are particularly compelling because there wereno statistical differences in residual white blood cell content (seeFIG. 7), residual hemoglobin content (see FIG. 8), red blood cellrecovery after filtration (see FIG. 9) or the volume of the whole bloodcell unit (FIG. 10) between the SOLX/DINCH bag sample and the AS-1/DEHPbag sample prior to storage. Thus, the results in FIGS. 3-6 were not dueto any differences in the whole blood or red blood cells themselves.Rather, the surprising results must be attributed to the fact that theadditive solution described herein provides such a beneficialenvironment in which to store RBCs, that it compensates for the lack ofbeneficial effect provided for by the DEHP plasticizer found in PVCblood storage containers widely used today. Most surprisingly of all, anon-limiting composition described herein, namely the SOLX additivesolution, in DINCH plasticized containers actually performed better thancurrently available storage compositions/plasticizer combinations.

The above examples are provided for illustrative purposes only andshould not be construed as limiting the scope of the present inventionas defined herein by the claims. Thus, it is within the scope of theclaimed invention to include PVC blood storage containers made withother non-DHEP plasticizers, as defined above.

Containers for storing the RBC compositions disclosed herein may bepermeable to oxygen or at least semi-permeable to oxygen. As shown inFIGS. 11 and 12, container 10 may include one or more container walls 12which define an interior chamber 15 for receiving the RBC composition20. In one embodiment, two sheets made of a plastic material are broughttogether and sealed along their peripheries 14 to form container 10.Other ways of making container 10 will be known to those of skill in theart and are within the scope of the present disclosure. As shown in FIG.8, container wall 12 includes an inner surface 13 which contacts theRBCs and an outer surface 17. In one embodiment, container wall may bemade of a single layer of a polymer material, such as PVC or non-PVCpolymer or polymer blend. In another embodiment, container wall 12 maybe made of a multiple sheet laminate wherein inner surface 13 is made ofone material and outer surface 17 is made of a different material.Container 10 may include one or more access ports 16 for connection withtubing 22, docking devices and the like to establish flow into and outfrom the interior chamber 15 of container 10.

In one embodiment, containers useful in the storage of RBCs as describedabove include container walls that are made in whole or at least in partof a plastic material that may include at least one or more polymericcompounds. The one or more plastic and/or polymeric compounds may beblended together and formed into flat sheets that are sealed together inthe manner described above. The polymeric material may be made from orotherwise include polyvinyl chloride (PVC) or one or more non-PVCpolyolefin homopolymers, copolymers or blends thereof. Examples ofsuitable non-PVC polyolefins include polypropylene, polyethylene,including ultra low density polyethylene (ULDPE) and very low densitypolyethylene (VLDPE). Other suitable compounds that may be used in theplastic materials of the containers or as part of the blend for makingthe plastic materials include ethylene vinyl acetate (EVA) and blockcopolymers such as Kraton®. Exemplary formulations and/or thepolyolefins, polyolefin blends or other polymeric compounds which areuseful, either alone or in combination, in the manufacture of containerssuitable for use in the RBC products of the present disclosure aredescribed in U.S. Pat. Nos. 5,026,347, 4,140,162, 5,849,843, and6,579,583, all of which are incorporated herein by reference in theirentireties.

As indicated above, the structure of the container or container wall mayinclude one, two or more layers. The layer formulations may include one,two, three or more components. These structures should be suitable forsterilization by appropriate means, such as steam, ionizing radiation orethylene oxide.

Structures suitable for steam sterilization should resist distortions tohigh temperatures up to 121° C. This typically requires incorporation ofmaterials with a melting peak of greater than 130° C. The preferredstructure of autoclavable material suitable for the invention willincorporate polypropylene homopolymer or copolymer at a level of 30% ormore in at least one of the layers to provide thermal resistance. Asuitable polypropylene copolymer is supplied by Total Petrochemicals(random copolymer 6575). However, thermal resistance can also beobtained by crosslinking a lower melting material. For example, a 28%vinyl acetate EVA can be crosslinked by ionizing radiation sufficientlyto withstand autoclave temperatures even though it has a melting pointof 76° C. Suitable materials include Arkema Evatane® 28-03 and CelaneseAteva® 2803. The preferred structure is highly flexible, having acomposite modulus of not more than 20,000 psi.

In some cases, it may be desirable for the structure to have radiofrequency (RF) response to enable heat sealing. This can be accomplishedby incorporating an RF responsive material such as described in U.S.Pat. No. 5,849,843.

Preferred structures for radiation sterilized applications willincorporate at least 30% of an ethylene based polymer (LDPE, LLDPE,ULDPE, VLDPE, EVA) in at least one of the layers. Structures ofpolypropylene copolymers and polypropylene polymers blended withelastomers such as Kraton® or ULDPE are also suitable for radiationsterilized applications. The preferred structure incorporates lowermodulus components in at least one of the layers to enhance flexibilityand toughness. These lower modulus components can be ultra low densitypolyethylene (ULDPE—typical density less than 0.90 Kg/L), very lowdensity polyethylene (VLDPE—typically density less than 0.925 Kg/L),ethylene vinyl acetate copolymers (EVA) with greater than 16% vinylacetate content, styrene butadiene terpolymers such as Kraton®. ULDPEmaterials are commercially available as Mitsui TAFMER®, Exxon MobilExact® and Dow Affinity®. EVA materials are available as Arkema Evatane®and Celanese Ateva®. These materials are incorporated at levelssufficient to obtain a composite modulus of less than 20,000 psi whilemaintaining resistance to distortion at temperatures greater than 121°C. for autoclaved applications. The disclosure of suitable non-PVCplastics set forth above is not meant to be exhaustive and it will beappreciated that other non-PVC plastics, polymers and blends thereof mayalso be used in the products and compositions of the present disclosure.Containers of the type described herein may have a container sheet(wall) thickness of between approximately 0.010 to 0.018 inches. Theymay include a non-smooth or any surface finish that minimizes sheetsticking. Typically, containers of the type described herein may have acontainer volume (i.e., interior chamber volume) of approximately 150 mlto 4 L.

As discussed above, containers useful in the methods, systems, andproducts disclosed herein may include PVC or be substantially free ofPVC. Thus, in one embodiment, with reference to FIG. 11, theformulations used to make container walls 12 of container 10 are atleast substantially free of polyvinylchloride (PVC). At the very least,surface 13 of container wall 12 is substantially free of PVC. In anembodiment where container 10 is made of a multiple sheet laminate thesheet providing inner surface 13 may be made substantially of a non-PVCmaterial while the sheet providing outer surface 17 may be made of adifferent material. More typically, however, the container wall 12 maybe made of a single sheet of a non-PVC polyolefin, as described above.

In some embodiments, even in containers where the walls 12 are madewithout PVC, some PVC may be present in small amounts. For example,ports 16 may include plasticized PVC. In any event, as used herein, theterms “substantially PVC-free” or “substantially free of PVC” refer tocontainers in which the walls that are in contact with the RBCcomposition, i.e., that part of the container that makes up a part ofthe storage environment, are made from a material that is free of PVC.

Containers suitable for use in the products, systems and methods of thepresent disclosure are at least substantially free of phthalateplasticizer, such as DEHP. This applies to containers where thepolymeric material is PVC plastic as well as where the polymericmaterial is a non-PVC plastic. In the case of a container that includesPVC, such container material will have to be plasticized due to thebrittle nature of PVC. As noted above, the plasticizer is anon-phthalate plasticizer. Non-phthalate plasticizers that may besuitable for use in the PVC containers described above include, forexample, triethylhexyltrimellitate (TEHTM) and the family of citrateesters, as described in U.S. Pat. No. 5,026,347.

Preferably, the PVC may be plasticized with 1,2-cyclohexane dicarboxylicacid diisononyl ester, known by its trade name, DINCH. In an embodimentwhere the polymer in the container material formulation is PVC, at least10%, by weight, of the formulation is preferably one or more preferablyhemolysis-suppressing, non-phthalate plasticizers such as DINCH or acitrate ester such as n-butyryltri-n-hexyl citrate. In an embodiment,PVC containers of the type described above may include approximately55-80%, by weight, PVC resin and approximately 20-45%, by weight, ofnon-phthalate plasticizer(s) wherein a preferred plasticizer is DINCH,and less than about 3.0% of stabilizers and lubricants. In a morespecific embodiment, containers of the type described above may includeapproximately 60-70% PVC resin, 20-35% DINCH plasticizer, approximately4-10% epoxidized oil and approximately 0.5-3.0% additionalco-stabilizers and lubricants. DINCH is available from, for example,BASF of Ludwigshafen, Germany.

In the case of non-PVC containers, such containers may likewise be freeof phthalate plasticizers but include non-phthalate plasticizer. Withrespect to either the PVC or the non-PVC containers, at least a portionof the container wall, i.e., the portion or surface that is in contactwith the RBCs during storage, is at least substantially free ofphthalate plasticizer. For example, with reference to FIG. 12, at leastinner surface 13 (or that portion of inner surface 13 that is in contactwith the RBCs) may be substantially free of phthalate plasticizer. Thus,the storage environment in which the RBCs reside is at leastsubstantially free of phthalate plasticizer. In a more specificembodiment, the storage environment in which the RBCs reside is at leastsubstantially free of phthalate plasticizer, includes a non-phthalateplasticizer and further includes a suitable storage solution (such asthose previously described).

Thus, for example, the PVC or non-PVC container (or more specifically,the container wall) is at least substantially free of a phthalateplasticizer but may include a non-phthalate plasticizer or extractableagent such as the citrate esters described in U.S. Pat. No. 5,026,347,or the DINCH plasticizer described above. Accordingly, suchnon-phthalate plasticizer(s) will be present in and part of the RBCstorage environment. As the containers disclosed herein are often partof a larger processing set that includes tubing, ports, membranes andconnectors in addition to being part of the storage environment, non-PVCand non-phthalate materials of the type described herein may also beused in the manufacture of such other processing set components.

In the embodiments described above, where the plastic container materialmay be PVC or a non-PVC composition, where no phthalate plasticizer oragent is included, and the material includes a single non-phthalateplasticizer (such as DINCH), a hypotonic, high pH storage media maypreferably be used in the red blood cell composition. RBC compositionsmay be stored in the substantially phthalate-free containers. The RBCcompositions stored in such containers may be stored for more than 21days, more than 35 days and up to at least 42 days or even up to atleast 49 days and/or 56 days, while maintaining acceptable storage cellfunction parameters (i.e., a level of ATP, 2,3-DPG, lactate). Inparticular, RBC compositions stored in the containers described aboveand that are substantially phthalate-free maintain hemolysis levelsbelow 1.0% and even below 0.8% at, for example, 42 days of storage.Similarly, the RBC compositions stored for at least about 42 days alsoinclude ATP, 2,3-DPG, lactate, potassium, phosphate levels that arecomparable to RBC compositions stored in plasticized PVC containers.

In another embodiment, the plastic composition may include two or moreplasticizers or extractable agents. The plastic container material maybe either PVC or the non-PVC materials described above. Likewise, thestructure of the container may also be as described above (single layeror multiple layers). Thus, in such an embodiment, the plasticcomposition may include a first extractable agent and a secondextractable agent. At least one of the agents is preferably anon-phthalate extractable agent/plasticizer (e.g., not DEHP). In someembodiments, none of the agents is DEHP. In an embodiment where both thefirst and the second extractable agents/plasticizers are non-phthalateplasticizers, one of the agents/plasticizers may be a non-phthalate,extractable agent that can suppress hemolysis such as, but not limitedto, the citrate ester n-butyryl-n-hexyl citrate (BTHC). More preferably,at least the first and second extractable agents or plasticizers areextractable agents or plasticizers, each effective in suppressinghemolysis in RBCs. Thus, in the embodiment where BTHC is one of suchextractable hemolysis-suppressing agents, the other of the at leastfirst or second agents or plasticizers may be a non-phthalateplasticizer, such as DINCH, which also is effective in suppressinghemolysis.

In another embodiment, the plastic composition may include a first andsecond extractable agent/plasticizer (wherein one of the first or secondagents/plasticizers is preferably BTHC) and a further or third agent orplasticizer. The further or third agent/plasticizer may likewise be anon-phthalate agent/plasticizer. The third plasticizer may be aplasticizer that is not readily extractable or marginally extractable,such as TEHTM or epoxidized oil (which also acts as a stabilizer).Alternatively or in addition, the third (or further) plasticizer may bemore readily extractable, such as the citrate ester acetyltri-nbutylcitrate (ATBC), which is also effective in suppressing hemolysis, orDINCH. Additional agents or plasticizers may further be included in theformulation of the containers described herein.

In a more specific embodiment, where the polymer in the containermaterial formulation is PVC, at least 10%, by weight, of the formulationincludes preferably two or more non-phthalate plasticizers wherein oneof the plasticizers is DINCH or a citrate ester such asn-butyryltri-n-hexyl citrate (BTHC). In an embodiment, containers of thetype described above may include approximately 55-80%, by weight, PVCresin and approximately 20-45%, by weight, of at least first and secondphthalate plasticizer(s) wherein a preferred plasticizer is BTHC and/orDINCH, and less than about 3.0% of stabilizers and lubricants. In a morespecific embodiment, containers of the type described above may includeapproximately 60-70% PVC resin, 15-30% DINCH plasticizer, 5-15% BTHC,approximately 4-10% epoxidized oil and approximately 0.5-3.0% ofadditional co-stabilizers and lubricants. DINCH is available from, forexample, BASF of Ludwigshafen, Germany.

In another embodiment, the plastic composition may include approximately55%-80%, by weight, PVC and approximately 20%-45% hemolysis-suppressingplasticizer/agent wherein, as a percentage of the overall composition,approximately 3-25% and more preferably 5%-15%, by weight, is a firstplasticizer/agent capable of suppressing hemolysis, such as BTHC. Inaccordance with the present disclosure, the plastic composition mayinclude 55%-80%, by weight, PVC and approximately 20%-45%, by weight, ofcombined hemolysis-suppressing plasticizer/agent wherein, as apercentage of the overall composition, approximately 5-15% is BTHC,2-12% epoxidized oil and 15-30% is one or more of ATBC, DINCH or otherextractable agents, each of which is effective to suppress hemolysis inred blood cells, with approximately 0.5-3.0% of additionalco-stabilizers and lubricants.

The compositions of the present disclosure may also include otheradditives such as anti-blocking and slip agents. Examples of suchanti-blocking and slip agents include derivatives of fatty acid andethylenediamine. More specifically, the agents may be longer chain fattyacids, containing 12 or longer hydrocarbon chains with or withoutmono-unsaturated carbon-carbon bonds, based daiamide withethylendiamine, such as n,n′-ethylene bissteararamide and n,n′-dioleoylethylenediamine. Commercially available compounds of the type describedabove and which may be used in the non-PVC, non-plasticized compositionsof the present disclosure include Acrawax and Glycolube, both availablefrom Lonza of Basel, Switzerland. The anti-blocking and/or slip agentsmay be coated onto the interior surface of the containers or otherwiseincorporated therein.

Compositions that include two or more preferably non-phthalateplasticizers or extractable agents are suitable for storing concentratedRBCs with an additive solution. In such applications, any additivesolution may be used.

In the embodiments where the container walls (or at least the innersurface(s) 13 of the walls) are made of a material completely free ofphthalate, some small trace amounts of phthalate may be present in thecontainer walls as a result of migration from adjoining or adjacentcontainers, from PVC tubing and/or the surrounding environmentgenerally. In addition, as described above, ports 16 may likewiseinclude PVC and as a result may include some plasticizer (includingDEHP). Nonetheless, the presence of some trace amounts of plasticizerattributable to migration from other containers or tubing, or present inthe plastic ports 16, is negligible and such containers are referred toherein as “substantially phthalate-free” or “substantially free ofphthalate.”

The embodiments of the invention described above are intended to bemerely exemplary; numerous variations and modifications will be apparentto those skilled in the art. All such variations and modifications areintended to be within the scope of the present invention as defined inany appended claims.

1. A combination product for storing red blood cells at about 1 to 6° C.for a storage period, the combination product comprising, consistingessentially of, or consisting of: (a) a container comprising, consistingessentially of, or consisting of a wall defining an interior chamberwherein at least a portion of the wall is made of polymeric materialcombined with a non-phthalate plasticizer; (b) a composition containedwithin said chamber, said composition comprising, consisting essentiallyof, or consisting of: adenine; dextrose; at least one non-metabolizablemembrane-protectant sugar; and a pH buffering system, wherein the pHbuffering system consists of a combination of physiologically acceptablebuffering agents including at least one agent providing bicarbonateanions, at least one agent providing phosphate anions, and at least oneagent providing sodium cations, wherein the aqueous composition issubstantially free of exogenously derived chloride ions, wherein the pHbuffering system is present in an amount sufficient for the compositionto be operable to maintain a pH of a red blood cell (RBC) suspension towhich the composition is added at a value sufficient to establish andmaintain during a storage period a reaction equilibrium in the red bloodcell that favors glycolysis over synthesis of 2,3-diphosphoglycerat(DPG) from 1,3-DPG, thereby generating a net gain in adenosinetriphosphate (ATP) with respect to the reaction equilibrium during thestorage period.
 2. The combination product of claim 1, whereincomposition contained within said chamber comprises, consists, orconsists essentially of: adenine at about 1 mM to about 3 mM; dextroseat about 20 mM to about 115 mM; disodium phosphate at about 4 mM toabout 15 mM; at least one non-metabolizable membrane-protectant sugar atabout 15 to about 60 mM; and a physiologically acceptable sodium salt atabout 20 mM to about 130 mM.
 3. The product of claim 1, wherein thephysiologically acceptable sodium salt is sodium bicarbonate.
 4. Theproduct of claim 1, wherein said polymeric material comprises polyvinylchloride.
 5. The product of claim 1, wherein said polymeric materialcomprises a non-PVC composition.
 6. The product of claim 1, wherein thenon-phthalate plasticizer is 1,2-cyclohexane dicaroxylic acid diisononylester.
 7. The product of claim 1, wherein the non-phthalate plasticizeris butyl-n-trihexyl-citrate (BTHC); di, (2, ethyl, hexyl) terephthalate(DENT); tri, (2-ethyl hexyl)trimellitate (TEHTM); or diisononylcyclohexane-1,2-dicarboxylate (Hexamoll™/DINCH).
 7. The product of claim1, wherein red blood cells stored in the product have a hemolysis levelthat is below 1.0% when the storage period at about 1 to 6° C. is atleast 6 weeks.
 8. The product of claim 1, wherein red blood cells storedin the product have a hemolysis level that is below 1.0% when thestorage period at about 1 to 6° C. is at least 8 weeks.
 9. The productof claim 1, wherein red blood cells stored in the product have ahemolysis level that is below 1.0% when the storage period at about 1 to6° C. is at least 10 weeks.
 10. The product of claim 1, wherein redblood cells stored in the product have a hemolysis level that is below1.0% when the storage period at about 1 to 6° C. is at least 12 weeks.11. The product of claim 1, wherein the composition is substantiallyfree of citrate.
 12. (canceled)
 13. The product of claim 1, whereinadenine is present in an amount of about 2 mM.
 14. (canceled)
 15. Theproduct of claim 1, wherein dextrose is present in an amount of betweenabout 60 mM to about 100 mM.
 16. The product of claim 1, whereindextrose is present in an amount of about 80 mM.
 17. (canceled)
 18. Theproduct of claim 1, wherein the unmetabolizable membrane-protectantsugar is present in an amount of between about 40 mM and about 60 mM.19. The product of claim 1 wherein the unmetabolizablemembrane-protectant sugar is present in an amount of about 55 mM. 20.The product of claim 1, wherein the unmetabolizable membrane-protectantsugar is mannitol.
 21. The product of claim 1, wherein the at least oneagent providing bicarbonate anions is sodium bicarbonate.
 22. (canceled)23. The product of claim 21, wherein the sodium bicarbonate is presentin an amount of between about 20 mM and about 40 mM.
 24. The product ofclaim 21, wherein the sodium bicarbonate is present in an amount ofabout 26 mM.
 25. The product of claim 1, wherein the at least one agentproviding phosphate anions is disodium phosphate.
 26. The product ofclaim 25, wherein the disodium phosphate is present in an amount ofbetween about 4 mM and about 20 mM.
 27. The product of claim 25, whereinthe disodium phosphate is present in an amount of between about 7 mM andabout 15 mM.
 28. The product of claim 25, wherein the disodium phosphateis present in an amount of about 12 mM.
 29. The product of claim 1,wherein the osmolarity of the composition is between about 210mOsmoles/liter and about 340 mOsmoles/liter.
 30. The product of claim 1,wherein the osmolarity of the composition is between about 220mOsmoles/liter and about 310 mOsmoles/liter.
 31. A method for thestorage of red blood cells comprising: (a) collecting a sample of wholeblood containing the RBCs to be stored and plasma in a blood storagecontainer, wherein the blood storage container comprises PVC and anon-DEHP plasticizer, (b) mixing the sample of collected whole bloodwith an anticoagulant solution, thereby forming a suspension ofcollected whole blood; (c) treating the suspension of collected wholeblood to deplete the plasma and concentrate the RBCs, thereby formingpacked RBCs; (d) mixing the packed RBCs with an amount of an aqueouscomposition sufficient to form a suspension of RBCs having about 35% toabout 80% RBCs by volume; (e) cooling the suspension of RBCs to about 1to about 6° C.; and (f) storing the cooled suspension of RBCs accordingto standard blood bank procedures, wherein the aqueous compositioncomprises, consists essentially of, or consists of: adenine; dextrose;at least one non-metabolizable membrane-protectant sugar; and a pHbuffering system, wherein the pH buffering system consists of acombination of physiologically acceptable buffering agents including atleast one agent providing bicarbonate anions, at least one agentproviding phosphate anions, and at least one agent providing sodiumcations, wherein the aqueous composition is substantially free ofexogenously derived chloride ions, wherein the pH buffering system ispresent in an amount sufficient for the composition to be operable tomaintain a pH of a red blood cell (RBC) suspension to which thecomposition is added at a value sufficient to establish and maintainduring a storage period a reaction equilibrium in the red blood cellthat favors glycolysis over synthesis of 2,3-diphosphoglycerat (DPG)from 1,3-DPG, thereby generating a net gain in adenosine triphosphate(ATP) with respect to the reaction equilibrium during the storageperiod.
 32. The method of claim 31, wherein the aqueous compositioncomprises, consists essentially of, or consists of: adenine at about 1mM to about 3 mM; dextrose at about 20 mM to about 115 mM; disodiumphosphate at about 4 mM to about 15 mM; at least one non-metabolizablemembrane-protectant sugar at about 15 to about 60 mM; and aphysiologically acceptable sodium salt at about 20 mM to about 130 mM.33. The method of claim 31, wherein the aqueous composition comprises,consists essentially of, or consists of adenine in an amount of about 1mM to about 3 mM, dextrose in an amount of from about 20 to about 115mM, unmetabolizable membrane-protectant sugar in an amount of about 15to about 60 mM, sodium bicarbonate in an amount from about 20 to about130 mM, and disodium phosphate in an amount of from about 4 to about 20mM.